Reverse-turn mimetics and method relating thereto

ABSTRACT

Conformationally constrained compounds that mimic the secondary structure of reverse-turn regions of biologically active peptides and proteins are disclosed. Such reverse-turn mimetic structures have utility over a wide range of fields, including use as diagnostic and therapeutic agents. Libraries containing the reverse-turn mimetic structures of this invention are also disclosed as well as methods for screening the same to identify biologically active members. The invention also relates to the use of such compounds for inhibiting or treating disorders modulated by Wnt-signaling pathway, such as cancer, especially colorectal cancer, restenosis associated with angioplasty, polycystic kidney disease, aberrant angiogenesis disease, rheumatoid arthritis disease, tuberous sclerosis complex, Alzheimer&#39;s disease, excess hair growth or loss, or ulcerative colitis.

CROSS-REFERENCES TO RELATED APPLICATIONS

This application is a continuation-in-part of U.S. patent applicationSer. No. 10/826,972, filed Apr. 16, 2004, which is acontinuation-in-part of co-pending U.S. patent application Ser. No.10/803,179 filed Mar. 17, 2004, which is a continuation-in-part of U.S.patent application Ser. No. 10/411,877 filed Apr. 9, 2003 (nowabandoned), which is a continuation-in-part of U.S. patent applicationSer. No. 10/087,443 filed Mar. 1, 2002 (now abandoned), which is acontinuation-in-part of U.S. patent application Ser. No. 09/976,470filed Oct. 12, 2001 (now abandoned). This application also claimspriority to PCT application serial No. PCT/KR02/01901 filed Oct. 11,2002. The disclosures of these applications are incorporated herein byreference in their entireties.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates generally to reverse-turn mimeticstructures and to a chemical library relating thereto. The inventionalso relates to applications in the treatment of medical conditions,e.g., cancer diseases, and pharmaceutical compositions comprising themimetics.

2. Description of the Related Art

Random screening of molecules for possible activity as therapeuticagents has occurred for many years and resulted in a number of importantdrug discoveries. While advances in molecular biology and computationalchemistry have led to increased interest in what has been termed“rational drug design”, such techniques have not proven as fast orreliable as initially predicted. Thus, in recent years there has been arenewed interest and return to random drug screening. To this end,particular strides having been made in new technologies based on thedevelopment of combinatorial chemistry libraries, and the screening ofsuch libraries in search for biologically active members.

In general, combinatorial chemistry libraries are simply a collection ofmolecules. Such libraries vary by the chemical species within thelibrary, as well as the methods employed to both generate the librarymembers and identify which members interact with biological targets ofinterest. While this field is still young, methods for generating andscreening libraries have already become quite diverse and sophisticated.For example, a recent review of various combinatorial chemical librarieshas identified a number of such techniques (Dolle, J. Com. Chem., 2(3):383-433, 2000), including the use of both tagged and untagged librarymembers (Janda, Proc. Natl. Acad. Sci. USA 91:10779-10785, 1994).

Initially, combinatorial chemistry libraries were generally limited tomembers of peptide or nucleotide origin. To this end, the techniques ofHoughten et al. illustrate an example of what is termed a “dual-definediterative” method to assemble soluble combinatorial peptide librariesvia split synthesis techniques (Nature (London) 354:84-86, 1991;Biotechniques 13:412-421, 1992; Bioorg. Med. Chem. Lett. 3:405-412,1993). By this technique, soluble peptide libraries containing tens ofmillions of members have been obtained. Such libraries have been shownto be effective in the identification of opioid peptides, such asmethionine- and leucine-enkephalin (Dooley and Houghten, Life Sci. 52,1509-1517, 1993), and a N-acylated peptide library has been used toidentify acetalins, which are potent opioid antagonists (Dooley et al.,Proc. Natl. Acad. Sci. USA 90:10811-10815, 1993. More recently, an allD-amino acid opioid peptide library has been constructed and screenedfor analgesic activity against the mu (“μ”) opioid receptor (Dooley etal, Science 266:2019-2022, 1994).

While combinatorial libraries containing members of peptide andnucleotide origin are of significant value, there is still a need in theart for libraries containing members of different origin. For example,traditional peptide libraries to a large extent merely vary the aminoacid sequence to generate library members. While it is well recognizedthat the secondary structures of peptides are important to biologicalactivity, such peptide libraries do not impart a constrained secondarystructure to its library members.

To this end, some researchers have cyclized peptides with disulfidebridges in an attempt to provide a more constrained secondary structure(Tumelty et al., J. Chem. Soc. 1067-68, 1994; Eichler et al., PeptideRes. 7:300-306, 1994). However, such cyclized peptides are generallystill quite flexible and are poorly bioavailable, and thus have met withonly limited success.

More recently, non-peptide compounds have been developed which moreclosely mimic the secondary structure of reverse-turns found inbiologically active proteins or peptides. For example, U.S. Pat. No.5,440,013 to Kahn and published PCT applications nos. WO94/03494,WO01/00210A1, and WO01/16135A2 to Kahn each disclose conformationallyconstrained, non-peptidic compounds, which mimic the three-dimensionalstructure of reverse-turns. In addition, U.S. Pat. No. 5,929,237 and itscontinuation-in-part U.S. Pat. No. 6,013,458, both to Kahn, discloseconformationally constrained compounds which mimic the secondarystructure of reverse-turn regions of biologically active peptides andproteins. The synthesis and identification of conformationallyconstrained, reverse-turn mimetics and their application to diseaseswere well reviewed by Obrecht (Advances in Med. Chem., 4, 1-68, 1999).

While significant advances have been made in the synthesis andidentification of conformationally constrained, reverse-turn mimetics,there remains a need in the art for small molecules which mimic thesecondary structure of peptides. There is also a need in the art forlibraries containing such members, as well as techniques forsynthesizing and screening the library members against targets ofinterest, particularly biological targets, to identify bioactive librarymembers.

The present invention also fulfills these needs, and provides furtherrelated advantages by providing confomationally constrained compoundswhich mimic the secondary structure of reverse-turn regions ofbiologically active peptides and proteins.

Wnt signaling pathway regulates a variety of processes including cellgrowth, oncogenesis, and development (Moon et al., 1997, Trends Genet.13, 157-162; Miller et al., 1999, Oncogene 18, 7860-7872; Nusse andVarmus, 1992, Cell 69, 1073-1087; Cadigan and Nusse, 1997, Genes Dev.11, 3286-3305; Peifer and Polakis, 2000 Science 287, 1606-1609; Polakis2000, Genes Dev. 14, 1837-1851). Wnt signaling pathway has beenintensely studied in a variety of organisms. The activation ofTCF4/β-catenin mediated transcription by Wnt signal transduction hasbeen found to play a key role in its biological functions (Molenaar etal., 1996, Cell 86:391-399; Gat et al., 1998 Cell 95:605-614; Orford etal., 1999 J. Cell. Biol. 146:855-868; Bienz and Clevers, 2000, Cell103:311-20).

In the absence of Wnt signals, tumor suppressor gene adenomatouspolyposis coli (APC) simultaneously interacts with the serine kinaseglycogen synthase kinase (GSK)-3β and β-catenin (Su et al., 1993,Science 262, 1734-1737: Yost et al., 1996 Genes Dev. 10, 1443-1454:Hayashi et al., 1997, Proc. Natl. Acad. Sci. USA, 94, 242-247: Sakanakaet al., 1998, Proc. Natl. Acad. Sci. USA, 95, 3020-3023: Sakanaka andWilliam, 1999, J. Biol. Chem 274, 14090-14093). Phosphorylation of APCby GSK-3β regulates the interaction of APC with β-catenin, which in turnmay regulate the signaling function of β-catenin (B. Rubinfeld et al.,Science 272, 1023, 1996). Wnt signaling stabilizes β-catenin allowingits translocation to the nucleus where it interacts with members of thelymphoid enhancer factor (LEF1)/T-cell factor (TCF4) family oftranscription factors (Behrens et al., 1996 Nature 382, 638-642: Hsu etal., 1998, Mol. Cell. Biol. 18, 4807-4818: Roose et all., 1999 Science285, 1923-1926).

Recently c-myc, a known oncogene, was shown to be a target gene forβ-catenin/TCF4-mediated transcription (He et al., 1998 Science 2811509-1512: Kolligs et al., 1999 Mol. Cell. Biol. 19, 5696-5706). Manyother important genes, including cyclin D1, and metalloproteinase, whichare also involved in oncogenesis, have been identified to be regulatedby TCF4/bata-catenin transcriptional pathway (Crawford et al., 1999,Oncogene 18, 2883-2891: Shtutman et al., 1999, Proc. Natl. Acad. Sci.USA., 11, 5522-5527: Tetsu and McCormick, 1999 Nature, 398, 422-426).

Moreover, overexpression of several downstream mediators of Wntsignaling has been found to regulate apoptosis (Moris et al., 1996,Proc. Natl. Acad. Sci. USA, 93, 7950-7954: He-et al., 1999, Cell 99,335-345: Orford et al, 1999 J. Cell. Biol., 146, 855-868: Strovel andSussman, 1999, Exp. Cell. Res., 253, 637-648). Overexpression of APC inhuman colorectal cancer cells induced apoptosis (Moris et al., 1996,Proc. Natl. Acad. Sci. USA., 93, 7950-7954), ectopic expression ofβ-catenin inhibited apoptosis associated with loss of attachment toextracellular matrix (Orford et al, 1999, J. Cell Biol. 146, 855-868).Inhibition of TCF4/β-catenin transcription by expression ofdominant-negative mutant of TCF4 blocked Wnt-1-mediated cell survivaland rendered cells sensitive to apoptotic stimuli such as anti-canceragent (Shaoqiong Chen et al., 2001, J. Cell. Biol., 152, 1, 87-96) andAPC mutation inhibits apoptosis by allowing constitutive survivinexpression, a well-known anti-apoptotic protein (Tao Zhang et al., 2001,Cancer Research, 62, 8664-8667).

Although mutations in the Wnt gene have not been found in human cancer,a mutation in APC or β-catenin, as is the case in the majority ofcolorectal tumors, results in inappropriate activation of TCF4,overexpression of c-myc and production of neoplastic growth (Bubinfeldet al, 1997, Science, 275, 1790-1792 Morin et al, 1997, Science, 275,1787-1790: Casa et al, 1999, Cell. Growth. Differ. 10, 369-376). Thetumor suppressor gene (APC) is lost or inactivated in 85% of colorectalcancers and in a variety of other cancers as well (Kinzler andVogelstein, 1996, Cell 87, 159-170). APC's principal role is that of anegative regulator of the Wnt signal transduction cascade. A centerfeature of this pathway involves the modulation of the stability andlocalization of a cytosolic pool of β-catenin by interaction with alarge Axin-based complex that includes APC. This interaction results inphosphorylation of β-catenin thereby targeting it for degradation.

CREB binding proteins (CBP)/p300 were identified initially in proteininteraction assays, first through its association with the transcriptionfactor CREB (Chrivia et al, 1993, Nature, 365, 855-859) and laterthrough its interaction with the adenoviral-transforming protein E1A(Stein et al., 1990, J. Viol., 64, 4421-4427: Eckner et al., 1994,Genes. Dev., 8, 869-884). CBP had a potential to participate in varietyof cellular functions including transcriptional coactivator function(Shikama et al., 1997, Trends. Cell. Biol., 7, 230-236: Janknecht andHunter, 1996, Nature, 383, 22-23). CBP/p300 potentiatesβ-catenin-mediated activation of the siamois promoter, a known Wnttarget (Hecht et al, 2000, EMBO J. 19, 8, 1839-1850). β-catenininteracts directly with the CREB-binding domain of CBP and β-cateninsynergizes with CBP to stimulate the transcriptional activation ofTCF4/β-catenin (Ken-Ichi Takemaru and Randall T. Moon, 2000 J. Cell.Biol., 149, 2, 249-254).

BRIEF SUMMARY OF THE INVENTION

From this background, it is seen that TCF4/β-catenin and CBP complex ofWnt pathway can be taken as target molecules for the regulation of cellgrowth, oncogenesis and apoptosis of cells, etc. Accordingly, thepresent invention addresses a need for compounds that blockTCF4/β-catenin transcriptional pathway by inhibiting CBP, and thereforecan be used for treatment of cancer, especially colorectal cancer.

In brief, the present invention is directed to a new type ofconformationally constrained compounds, which mimic the secondarystructure of reverse-turn regions of biologically active peptides andproteins. This invention also discloses libraries containing suchcompounds, as well as the synthesis and screening thereof.

The compounds of the present invention have the following generalformula (I):

wherein A is —(CHR₃)— or —(C═O)—, B is —(CHR₄)— or —(C═O)—, D is—(CHR₅)— or —(C═O)—, E is -(ZR₆)— or —(C═O)—, G is —(XR₇)_(n)—,—(CHR₇)—(NR₈)—, —(C═O)—(XR₉)—, or —(C═O)—, W is —Y(C═O)—, —(C═O)NH—,—(SO₂)— or is absent, Y is oxygen, sulfur, or —NH—, X and Z isindependently nitrogen or CH, n=0 or 1; and R₁, R₂, R₃, R₄, R₅, R₆, R₇,R₈ and R₉ are the same or different and independently selected from anamino acid side chain moiety or derivative thereof, the remainder of themolecule, a linker and a solid support, and stereoisomers thereof.

In an embodiment wherein A is —CHR₃)—, B is —(C═O)—, D is —(CHR₅)—, E is—(C═O)—, and G is —XR₇)_(n)—, the compounds of this invention have thefollowing formula (II):

wherein W, X, Y and n are as defined above, and R₁, R₂, R₃, R₅ and R₇are as defined in the following detailed description.

In an embodiment wherein A is —(C═O)—, B is —(CHR₄)—, D is —(C═O)—, E is-(ZR₆)—, and G is —(C═O)—(XR₉)—, the compounds of this invention havethe following formula (III):

wherein W, X and Y are as defined above, Z is nitrogen or CH (with theproviso that when Z is CH, then X is nitrogen), and R₁, R₂, R₄, R₆ andR₉ are as defined in the following detailed description.

In an embodiment wherein A is —(C═O)—, B is —(CHR₄)—, D is —(C═O)—, E is-(ZR₆)—, and G is (XR₇)_(n)—, the compounds of this invention have thefollowing general formula (IV):

wherein W, Y and n are as defined above, Z is nitrogen or CH (when Z isnitrogen, then n is zero, and when Z is CH, then X is nitrogen and n isnot zero), and R₁, R₂, R₄, R₆ and R₇, are as defined in the followingdetailed description.

In certain embodiments, the compounds of this invention have thefollowing general formula (VI):

wherein R_(a) is a phenyl group; a substituted phenyl group having oneor more substituents wherein the one or more substituents areindependently selected from one or more of amino, amidino, guanidino,hydrazino, amidazonyl, C₁₋₄alkylamino, C₁₋₄dialkylamino, halogen,perfluoro C₁₋₄alkyl, C₁₋₄alkyl, C₁₋₃alkoxy, nitro, carboxy, cyano,sulfuryl, and hydroxyl groups; a benzyl group; a substituted benzylgroup with one or more substituents where the one or more substituentsare independently selected from one or more of amino, amidino,guanidino, hydrazino, amidazonyl, C₁₋₄alkylamino, C₁₋₄dialkylamino,halogen, perfluoro C₁₋₄alkyl, C₁₋₃alkoxy, nitro, carboxy, cyano,sulfuryl, and hydroxyl group; or a bicyclic aryl group having 8 to 11ring members, which may have 1 to 3 heteroatoms selected from nitrogen,oxygen or sulfur; R_(b) is a monocyclic aryl group having 5 to 7 ringmembers, which may have 1 to 2 heteroatoms selected from nitrogen,oxygen or sulfur, and aryl ring in the compound may have one or moresubstituents selected from a group consisting of halide, hydroxy, cyano,lower alkyl, and lower alkoxy groups; R_(c) is a saturated orunsaturated C₁₋₆alkyl, C₁₋₆alkoxy, perfluoro C₁₋₆alkyl group; and X₁,X₂, and X₃ may be the same or different and independently selected fromhydrogen, hydroxyl, and halide.

The present invention is also related to prodrugs using the librariescontaining one or more compounds of formula (I). A prodrug is typicallydesigned to release the active drug in the body during or afterabsorption by enzymatic and/or chemical hydrolysis. The prodrug approachis an effective means of improving the oral bioavailability or i.v.administration of poorly water-soluble drugs by chemical derivatizationto more water-soluble compounds. The most commonly used prodrug approachfor increasing aqueous solubility of drugs containing a hydroxyl groupis to produce esters containing an ionizable group; e.g., phosphategroup, carboxylate group, alkylamino group (Fleisher et al., AdvancedDrug Delivery Reviews, 115-130, 1996; Davis et al., Cancer Res.,7247-7253, 2002, Golik et al., Bioorg. Med. Chem. Lett., 1837-1842,1996).

In certain embodiments, the prodrugs of the present invention have thefollowing general formula (VII):(VI)—Y—R₁₀wherein (VI) is general formula (VI) as described above; Y is oxygen,sulfur, or nitrogen of a group selected from R_(a), R_(b), R_(c), X₁, X₂and X₃;

R₁₀ is phosphate, hemisuccinate, hemimalate,phosphoryloxymethyloxycarbonyl, dimethylaminoacetate,dimethylaminoalkylcarbamates, hydroxyalkyls, amino acid, glycosyl,substituted or unsubstituted piperidine oxycarbonyl, or a salt thereof;and wherein the prodrugs are capable of serving as a substrate for aphosphatase or a carboxylase and are thereby converted to compoundshaving general formula (VI).

In some embodiments, R₁₀ of the general formula (VII) is not an aminoacid group or a phospho-amino acid group.

The present invention is also directed to libraries containing one ormore compounds of formula (I) above, as well as methods for synthesizingsuch libraries and methods for screening the same to identifybiologically active compounds. Compositions containing a compound ofthis invention in combination with a pharmaceutically acceptable carrieror diluent are also disclosed.

The present invention is also related to methods for identifying abiologically active compound using the libraries containing one or morecompound of formula (I). In a related aspect, the present inventionprovides a method for performing a binding assay, comprising (a)providing a composition comprising a first co-activator and aninteracting protein, said first co-activator comprising a binding motifof LXXLL, LXXLI or FXXFF wherein X is any amino acid; (b) combining thefirst co-activator and the interacting protein with a test compound; and(c) detecting alteration in binding between the first co-activator andthe interacting protein in the presence of the compound having generalformula (I).

The present invention also provides methods for preventing or treatingdisorders associated with Wnt signaling pathway. Disorders that may betreated or prevented using a compound or composition of the presentinvention include tumor or cancer (e.g., KSHV-associated tumor),restenosis associated with angioplasty, polycystic kidney disease,aberrant angiogenesis disease, rheumatoid arthritis disease, ulcerativecolitis, tuberous sclerosis complex, hair loss, and Alzheimer's disease.Such methods comprise administering to a subject in need thereof acompound or composition of the present invention in an amount effectiveto achieve the desired outcome.

In a related aspect, the present invention further provides methods forpromoting neurite outgrowth, differentiation of a neural stem cell, andapoptosis in cancer cells. Such methods comprise administering toappropriate cells a compound or composition of the present invention inan amount effective to achieve the desired outcome.

These and other aspects of this invention will be apparent uponreference to the attached figure and following detailed description. Tothis end, various references are set forth herein, which describe inmore detail certain procedures, compounds and/or compositions, and areincorporated by reference in their entirety.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 provides a general synthetic scheme for preparing reverse-turnmimetics of the present invention.

FIG. 2 provides a general synthetic scheme for preparing reverse-turnmimetics of the present invention.

FIG. 3 shows a graph based on the measurement of IC₅₀ for Compound A ofthe present invention using SW480 cells, wherein cell growth inhibitionon SW480 cells was measured at various concentrations of Compound Aprepared in Example 4 to obtain the IC₅₀ value. Specifically, the degreeof inhibition in firefly and renilla luciferase activities by Compound Awas determined. As a result, the IC₅₀ of Compound A against SW480 cellgrowth was found as disclosed in Table 4. Detailed procedures are thesame as disclosed in Example 6.

FIG. 4. PC-12 cells were cultured on coated dishes, and differentiatedfor 10 days in 50 ng/ml nerve growth factor (NGF) (as described inExample 7). (A, B) Vector-transfected PC-12 cells (A) and PC-12 cellsoverexpressing wt PS-1 (B) exhibit extensive neurite outgrowth after 10days in NGF. (C) PC-12 cells expressing mutant PS-1/L286V do not displaysignificant neurites under the same culture conditions. (D, E)Immunofluorescence analysis of GAP-43 (as described in Example 7), amolecular marker of neurite outgrowth, demonstrates intense staining forGAP-43 in the neurites (D) of vector-transfected and overexpressingPS-1/WT in PC-12 cells (E). (F) Lack of neurite outgrowth corresponds toweak GAP-43 immunostaining in the mutant cells. Data represent at leasttwo independent experiments. (G) Differentiated cells were transfectedwith, Topflash, a TCF/β-catenin reporter construct. Cells were lysed,and luciferase activity measured 6 hours post-transfection (as describedin Example 7). Data represent the mean of three independent experiments(±SD). Asterisk indicate P<0.05.

FIG. 5. Compound D phenotypically corrects deficient neuronaldifferentiation in PC-12 overexpressing mutant PS-1/L286V cells. Mutantcells were exposed to 10 μM Compound D, in addition to NGF, during thedifferentiation period (Misner et al., Proc. Natl. Acad. Sci. USA 98,11714 (2001)). (A) Neurite elongation and extension are observed inPC-12 cells overexpressing PS-1/L286V upon treatment with Compound D.(B) GAP-43 (green) is significantly elevated in the mutant cells, and isseen in the neurites. (C) Quantitation of neurite outgrowth in PC-12cells. Number of mutant cells with neurite lengths greater than two celldiameters was less than 10% that of the vector-transfected andoverexpressing PS-1/WT in PC-12 cells. Number of mutant PS-1/L286V cellsthat had the defined neurite lengths was significantly increased, aftertreatment with 10 μM Compound D. The results are the average (±SD) ofthree independent determinations. Asterisk indicate P<0.05.

FIG. 6. Ephrin B2 (EphB2) receptor expression. Immunofluorescenceanalysis and RT-PCR were performed to detect EphB2 receptor expression(as described in Example 7). (A, B) EphB2 receptors are clearlydemonstrated in neurites of vector-transfected and overexpressingPS-1/WT cells. The intensity of staining correlates with the highexpression level. (C) In contrast, PS-1/L286V PC-12 cells have markedlyreduced EphB2 receptor expression. (D) Treatment of mutant cells withCompound D leads to increased EphB2 receptor expression, which isfocused at points of neurite outgrowth. (E) Expression of EphB2 receptorhas previously been shown to be transcriptionally regulated (Guo et al.,J. Neurosci. 17, 4212 (1997).). Lane 1, vector-transfected PC-12 cells,lane 2, overexpressing PS-1/WT-cells, lane 3, overexpressing mutantPS-1/L286V cells, lane 4, mutant cells treated with Compound D. RT-PCRanalysis indicates message for EphB2 receptor in cells overexpressingmutant PS-1/L286V is decreased compared to those in both thevector-transfected and overexpressing wt PS-1 PC-12 cells. Treatmentwith 10 μM Compound D upregulates EphB2 message. GAPDH is used aninternal control.

FIG. 7. A. Compound D arrests cells in G₁. FACS analysis was performedon SW480 (lower panel) and HCT116 (upper panel) cells treated for 24hours with either Compound D (25 μM) (right) or control (0.5% DMSO(left). 5.5×10⁶ cells were fixed and stained with propidium iodide (PI).B. Compound D selectively activates caspases in colon carcinoma celllines. SW480 and HCT116 (left graph) cells (105) along with the normalcolonocytes CCD18Co (right graph) were treated with either control (0.5%DMSO) or Compound D (25 μM). 24 hours post treatment, cells were lysedand the caspase-3/7 enzymatic activities were measured. Relativefluorescence units (RFU) were calculated by subtracting the unit valuesof the blank (control, without cells) from the treated samples (CompoundD or control) and plotted.

FIG. 8. Compound D reduces colony growth in soft agar in a dosedependent manner. Increasing concentrations of 5-fluorouracil (5-FU)(0.5-32 μM) and Compound D (0.25-5 μM) were added to SW480 (5000cells/well) of triplicate wells. Cells were washed and suspended in softagar growth medium. The number of colonies after 8 days (colonies over60 μM diameter) were counted and plotted against the compoundconcentration. Mean±SE of three determinations is indicated. The colonynumber of control in the absence of the compound was 1,637±71.

FIG. 9. A. Compound C reduces tumor-growth in nude mouse model. B.Compound C slightly reduces body weight in nude mouse model.

FIG. 10. The survivin transcriptional activity is upregulated by Wnt1,but knout-down by Compound D. Percent luciferase activities weremeasured in wildtype, CBP+/−, and p300+/−3T3 cells in the absence ofWnt1 and Compound D, or in the presence of Wnt1, Compound D or both.

FIG. 11. Compound A (right graph) and Compound D (left graph) inhibitthe activity of a survivin luciferase reporter in SW480 cells. Theluciferase activities under the control of the survivin promoter weremeasured in SW480 cells treated with compound A or Compound D at variousconcentrations.

FIG. 12. RT-PCR analsis indicates that Compound D treatment decreasesthe expression level of the survivin gene.

FIG. 13. Compound D decreases the association of various proteins withthe survivin promoter. ChIP assays on SW480 cells treated with eitherCompound D (25 μM) or control (0.5% DMSO) for 18 hours were performed.

FIG. 14. Compound D decreases survivin expression at the translationallevel. A. Western blot analysis of extracts of cells treated withvehicle (0.5% DMSO) alone, 10 μM or 25 μM Compound D, or 5 μM 5-FU wasperformed using survivin 6E4 monoclonal antibody (Cell SignalingTechnolgy). B. Survivin immunofluorescence microscopy. Cultured cancercells were fixed and stained with anti-survivin green. C. Survivinimmunofluorescence microscopy. SW480 cells treated with Compound D werefixed and stained with anti-survivin green.

FIG. 15. Compound D activates the caspase 3 activity (but not thecaspase 2 activity) via suppression of the survivin expression. Culturedcells with or without transfection of a construct containing thesurvivin gene were treated with stausporine (0.5 μM), Compound D (2.5 μMor 5.0 μM), or both. The caspase 2 and caspase 3 activities in thesecells were measured.

FIG. 16. Compound D promotes cell death via suppression of the survivinexpression. Cultured cancer cells with or without transfection of aconstruct containing the survivin gene were treated with stausporine(0.5 μM), Compound D (5.0 μM), or both. The cell death of these cellswas measured.

FIG. 17. Compound D increases the number of cells in G₀. Cultured cancercells with or without transfection of a construct containing thesurvivin gene were treated with stausporine (0.5 μM), Compound D (5 μM),or both. FACS analysis was performed on these cells and the percentagesof cells in G₀ are indicated.

FIG. 18 shows the changes of concentrations of prodrug A and its parentcompound in mouse plasma with the increase of time after i.v. bolusinjection of prodrug A. Square: parent compound; Diamond: prodrug A.

DETAILED DESCRIPTION OF THE INVENTION

The present invention is directed to conformationally constrainedcompounds that mimic the secondary structure of reverse-turn regions ofbiological peptide and proteins (also referred to herein as“reverse-turn mimetics”, and is also directed to chemical librariesrelating thereto.

The reverse-turn mimetic structures of the present invention are usefulas bioactive agents, including (but not limited to) use as diagnostic,prophylactic and/or therapeutic agents. The reverse-turn mimeticstructure libraries of this invention are useful in the identificationof bioactive agents having such uses. In the practice of the presentinvention, the libraries may contain from tens to hundreds to thousands(or greater) of individual reverse-turn structures (also referred toherein as “members”).

In one aspect of the present invention, a reverse-turn mimetic structureis disclosed having the following formula (I):

wherein A is —(CHR₃)— or —(C═O)—, B is —(CHR₄)— or —(C═O)—, D is—(CHR₅)— or —(C═O)—, E is -(ZR₆)— or —(C═O)—, G is —(XR₇)_(n)—,—(CHR₇)—(NR₈)—, —(C═O)—(XR₉)—, or —(C═O)—, W is —Y(C═O)—, —(C═O)NH—,—(SO₂)— or nothing, Y is oxygen, sulfur, or —NH—, X and Z isindependently nitrogen or CH, n=0 or 1; and R₁, R₂, R₃, R₄, R₅, R₆, R₇,R₈ and R₉ are the same or different and independently selected from anamino acid side chain moiety or derivative thereof, the remainder of themolecule, a linker and a solid support, and stereoisomers thereof.

In one embodiment, R₁, R₂, R₃, R₄, R₅, R₆, R₇, R₈ and R₉ areindependently selected from the group consisting of aminoC₂₋₅alkyl,guanidineC₂₋₅alkyl, C₁₋₄alkylguanidinoC₂₋₅alkyl,diC₁₋₄alkylguanidinoC₂₋₅alkyl, amidinoC₂₋₅alkyl,C₁₋₄alkylamidinoC₂₋₅alkyl, diC₁₋₄alkylamidinoC₂₋₅alkyl, C₁₋₃alkoxy,phenyl, substituted phenyl (where the substituents are independentlyselected from one or more of amino, amidino, guanidino, hydrazino,amidazonyl, C₁₋₄alkylamino, C₁₋₄dialkylamino, halogen, perfluoroC₁₋₄alkyl, C₁₋₄alkyl, C₁₋₃alkoxy, nitro, carboxy, cyano, sulfuryl orhydroxyl), benzyl, substituted benzyl (where the substituents on thebenzyl are independently selected from one or more of amino, amidino,guanidino, hydrazino, amidazonyl, C₁₋₄alkylamino, C₁₋₄dialkylamino,halogen, perfluoro C₁₋₄alkyl, C₁₋₃alkoxy, nitro, carboxy, cyano,sulfuryl or hydroxyl), naphthyl, substituted naphthyl (where thesubstituents are independently selected from one or more of amino,amidino, guanidino, hydrazino, amidazonyl, C₁₋₄alkylamino,C₁₋₄dialkylamino, halogen, perfluoro C₁₋₄alkyl, C₁₋₄alkyl, C₁₋₃alkoxy,nitro, carboxy, cyano, sulfuryl or hydroxyl), bis-phenyl methyl,substituted bis-phenyl methyl (where the substituents are independentlyselected from one or more of amino, amidino, guanidino, hydrazino,amidazonyl, C₁₋₄alkylamino, C₁₋₄dialkylamino, halogen, perfluoroC₁₋₄alkyl, C₁₋₄alkyl, C₁₋₃alkoxy, nitro, carboxy, cyano, sulfuryl orhydroxyl), pyridyl, substituted pyridyl, (where the substituents areindependently selected from one or more of amino amidino, guanidino,hydrazino, amidazonyl, C₁₋₄alkylamino, C₁₋₄dialkylamino, halogen,perfluoro C₁₋₄alkyl, C₁₋₄alkyl, C₁₋₃alkoxy, nitro, carboxy, cyano,sulfuryl or hydroxyl), pyridylC₁₋₄alkyl, substituted pyridylC₁₋₄alkyl(where the pyridine substituents are independently selected from one ormore of amino, amidino, guanidino, hydrazino, amidazonyl,C₁₋₄alkylamino, C₁₋₄dialkylamino, halogen, perfluoro C₁₋₄alkyl,C₁₋₄alkyl, C₁₋₃alkoxy, nitro, carboxy, cyano, sulfuryl or hydroxyl),pyrimidylC₁₋₄alkyl, substituted pyrimidylC₁₋₄alkyl (where the pyrimidinesubstituents are independently selected from one or more of amino,amidino, guanidino, hydrazino, amidazonyl, C₁₋₄alkylamino,C₁₋₄dialkylamino, halogen, perfluoro C₁₋₄alkyl, C₁₋₄alkyl, C₁₋₃alkoxy,nitro, carboxy, cyano, sulfuryl or hydroxyl), triazin-2-yl-C₁₋₄alkyl,substituted triazin-2-yl-C₁₋₄alkyl (where the triazine substituents areindependently selected from one or more of amino, amidino, guanidino,hydrazino, amidazonyl, C₁₋₄alkylamino, C₁₋₄dialkylamino, halogen,perfluoro C₁₋₄alkyl, C₁₋₄alkyl, C₁₋₃alkoxy, nitro, carboxy, cyano,sulfuryl or hydroxyl), imidazoC₁₋₄alkyl, substituted imidazol C₁₋₄alkl(where the imidazole sustituents are independently selected from one ormore of amino, amidino, guanidino, hydrazino, amidazonyl,C₁₋₄alkylamino, C₁₋₄dialkylamino, halogen, perfluoro C₁₋₄alkyl,C₁₋₄alkyl, C₁₋₃alkoxy, nitro, carboxy, cyano, sulfuryl or hydroxyl),imidazolinylC₁₋₄alkyl, N-amidinopiperazinyl-N—C₀₋₄alkyl,hydroxyC₂₋₅alkyl, C₁₋₅alkylaminoC₂₋₅alkyl, hydroxyC₂₋₅alkyl,C₁₋₅alkylaminoC₂₋₅alkyl, C₁₋₅dialkylaminoC₂₋₅alkyl,N-amidinopiperidinylC₁₋₄alkyl and 4-aminocyclohexylC₀₋₂alkyl.

In one embodiment, R₁, R₂, R₆ of E, and R₇, R₈ and R₉ of G are the sameor different and represent the remainder of the compound, and R₃ of A,R₄ of B or R₅ of D is selected from an amino acid side chain moiety orderivative thereof. As used herein, the term “remainder of the compound”means any moiety, agent, compound, support, molecule, linker, aminoacid, peptide or protein covalently attached to the reverse-turn mimeticstructure at R₁, R₂, R₅, R₆, R₇, R₈ and/or R₉ positions. This term alsoincludes amino acid side chain moieties and derivatives thereof.

In another embodiment R₃ of A, R₅ of D, R₆ of E, and R₇, R₈, and R₉ of Gare the same or different and represent the remainder of the compound,while one or more of, and in one aspect all of, R₁, R₂ and R₄ of Brepresent an amino acid sidechain. In this case, the term “remainder ofthe compound” means any moiety, agent, compound, support, molecule,linker, amino acid, peptide or protein covalently attached to thereverse-turn mimetic structure at R₃, R₅, R₆, R₇, R₈ and/or R₉positions. This term also includes amino acid side chain moieties andderivatives thereof.

As used herein, the term “remainder of the compound” means any moiety,agent, compound, support, molecule, atom, linker, amino acid, peptide orprotein covalently attached to the reverse-turn mimetic structure. Thisterm also includes amino acid side chain moieties and derivativesthereof. In one aspect of the invention, any one or more of the R₁, R₂,R₃, R₄, R₅, R₆, R₇, R₈ and/or R₉ positions may represent the remainderof the compound. In one aspect of the invention, one or more of R₁, R₂and R₄ represents an amino acid side chain moiety or a derivativethereof.

As used herein, the term “amino acid side chain moiety” represents anyamino acid side chain moiety present in naturally occurring proteinsincluding (but not limited to) the naturally occurring amino acid sidechain moieties identified in Table 1. Other naturally occurring aminoacid side chain moieties of this invention include (but are not limitedto) the side chain moieties of 3,5-dibromotyrosine, 3,5-diiodotyrosine,hydroxylysine, γ-carboxyglutamate, phosphotyrosine and phosphoserine. Inaddition, glycosylated amino acid side chains may also be used in thepractice of this invention, including (but not limited to) glycosylatedthreonine, serine and asparagine.

TABLE 1 Amino Acid Side Chain Moiety Amino Acid —H Glycine —CH₃ Alanine—CH(CH₃)₂ Valine —CH₂CH(CH₃)₂ Leucine —CH(CH₃)CH₂CH₃ Isoleucine—(CH₂)₄NH₃ ⁺ Lysine —(CH₂)₃NHC(NH₂)NH₂ ⁺ Arginine

Histidine —CH₂COO⁻ Aspartic acid —CH₂CH₂COO Glutamic acid —CH₂CONH₂Asparagine —CH₂CH₂CONH₂ Glutamine

Phenylalanine

Tyrosine

Tryptophan —CH₂SH Cysteine —CH₂CH₂SCH₃ Methionine —CH₂OH Serine—CH(OH)CH₃ Threonine

Proline

Hydroxyproline

In addition to naturally occurring amino acid side chain moieties, theamino acid side chain moieties of the present invention also includevarious derivatives thereof. As used herein, a “derivative” of an aminoacid side chain moiety includes modifications and/or variations tonaturally occurring amino acid side chain moieties. For example, theamino acid side chain moieties of alanine, valine, leucine, isoleucineand phenylalanine may generally be classified as lower chain alkyl,aryl, or arylalkyl moieties. Derivatives of amino acid side chainmoieties include other straight chain or branched, cyclic or noncyclic,substituted or unsubstituted, saturated or unsaturated lower chainalkyl, aryl or arylalkyl moieties.

As used herein, “lower chain alkyl moieties” contain from 1-12 carbonatoms, “lower chain aryl moieties” contain from 6-12 carbon atoms and“lower chain aralkyl moieties” contain from 7-12 carbon atoms. Thus, inone embodiment, the amino acid side chain derivative is selected from aC₁₋₁₂ alkyl, a C₆₋₁₂ aryl and a C₇₋₁₂ arylalkyl, and in a more preferredembodiment, from a C₁₋₇ alkyl, a C₆₋₁₀ aryl and a C₇₋₁₁ arylalkyl.

Amino side chain derivatives of this invention further includesubstituted derivatives of lower chain alkyl, aryl, and arylalkylmoieties, wherein the substituent is selected from (but is not limitedto) one or more of the following chemical moieties: —OH, —OR, —COOH,—COOR, —CONH₂, —NH₂, —NHR, —NRR, —SH, —SR, —SO₂R, —SO₂H, —SOR andhalogen (including F, Cl, Br and I), wherein each occurrence of R isindependently selected from straight chain or branched, cyclic ornoncyclic, substituted or unsubstituted, saturated or unsaturated lowerchain alkyl, aryl and aralkyl moieties. Moreover, cyclic lower chainalkyl, aryl and arylalkyl moieties of this invention includenaphthalene, as well as heterocyclic compounds such as thiophene,pyrrole, furan, imidazole, oxazole, thiazole, pyrazole, 3-pyrroline,pyrrolidine, pyridine, pyrimidine, purine, quinoline, isoquinoline andcarbazole. Amino acid side chain derivatives further include heteroalkylderivatives of the alkyl portion of the lower chain alkyl and aralkylmoieties, including (but not limited to) alkyl and aralkyl phosphonatesand silanes.

Representative R₁, R₂, R₃, R₄, R₅, R₆, R₇, R₈ and R₉ moietiesspecifically include (but are not limited to) —OH, —OR, —COR, —COOR,—CONH₂, —CONR, —CONRR, —NH₂, —NHR, —NRR, —SO₂R and —COSR, wherein eachoccurrence of R is as defined above.

In a further embodiment, and in addition to being an amino acid sidechain moiety or derivative thereof (or the remainder of the compound inthe case of R₁, R₂, R₃, R₅, R₆, R₇, R₈ and R₉), R₁, R₂, R₃, R₄, R₅, R₆,R₇, R₈ or R₉ may be a linker facilitating the linkage of the compound toanother moiety or compound. For example, the compounds of this inventionmay be linked to one or more known compounds, such as biotin, for use indiagnostic or screening assay. Furthermore, R₁, R₂, R₃, R₄, R₅, R₆, R₇,R₈ or R₉ may be a linker joining the compound to a solid support (suchas a support used in solid phase peptide synthesis) or alternatively,may be the support itself. In this embodiment, linkage to another moietyor compound, or to a solid support, is preferable at the R₁, R₂, R₇ orR₈, or R₉ position, and more preferably at the R₁ or R₂ position.

In the embodiment wherein A is —CHR₃)—, B is —(C═O)—, D is —(CHR₅)—, Eis —(C═O)—, and G is —XR₇)_(n)—, the reverse turn mimetic compound ofthis invention has the following formula (II):

wherein R₁, R₂, R₃, R₅, R₇, W, X and n are as defined above. In apreferred embodiment, R₁, R₂ and R₇ represent the remainder of thecompound, and R₃ or R₅ is selected from an amino acid side chain moiety.

In the embodiment wherein A is —(C═O)—, B is —(CHR₄)—, D is —(C═O)—, Eis -(ZR₆)—, G is —(C═O)—(XR₉)—, the reverse turn mimetic compound ofthis invention has the following general formula (III):

wherein R₁, R₂, R₄, R₆, R₉, W and X are as defined above, Z is nitrogenor CH (when Z is CH, then X is nitrogen). In a preferred embodiment, R₁,R₂, R₆ and R₉ represent the remainder of the compound, and R₄ isselected from an amino acid side chain moiety.

In a more specific embodiment wherein A is —(C═O)—, B is —(CHR₄)—, D is—(C═O)—, E is -(ZR₆)—, and G is (XR₇)_(n)—, the reverse turn mimeticcompound of this invention has the following formula (IV):

wherein R₁, R₂, R₄, R₆, R₇, W, X and n are as defined above, and Z isnitrogen or CH (when Z is nitrogen, then n is zero, and when Z is CH,then X is nitrogen and n is not zero). In a preferred embodiment, R₁,R₂, R₆ and R₇ represent the remainder of the compound, and R₄ isselected from an amino acid side chain moiety. In one aspect, R₆ or R₇is selected from an amino acid side chain moiety when Z and X are bothCH.

These compounds may be prepared by utilizing appropriate startingcomponent molecules (hereinafter referred to as “component pieces”).Briefly, in the synthesis of reverse-turn mimetic structures havingformula (I), first and second component pieces are coupled to form acombined first-second intermediate, if necessary, third and/or fourthcomponent pieces are coupled to form a combined third-fourthintermediate (or, if commercially available, a single third intermediatemay be used), the combined first-second intermediate and third-fourthintermediate (or third intermediate) are then coupled to provide afirst-second-third-fourth intermediate (or first-second-thirdintermediate) which is cyclized to yield the reverse-turn mimeticstructures of this invention. Alternatively, the reverse-turn mimeticstructures of formula (I) may be prepared by sequential coupling of theindividual component pieces either stepwise in solution or by solidphase synthesis as commonly practiced in solid phase peptide synthesis.

Specific component pieces and the assembly thereof to prepare compoundsof the present invention are illustrated in FIG. 1. For example, a“first component piece” may have the following formula S1:

wherein R₂ is as defined above, and R is a protective group suitable foruse in peptide synthesis, where this protection group may be joined to apolymeric support to enable solid-phase synthesis. Suitable R groupsinclude alkyl groups and, in a preferred embodiment, R is a methylgroup. In FIG. 1, one of the R groups is a polymeric (solid) support,indicated by “Pol” in the Figure. Such first component pieces may bereadily synthesized by reductive amination of H₂N—R₂ with CH(OR)₂—CHO,or by a displacement reaction between H₂N—R₂ and CH(OR)₂—CH₂-LG (whereinLG refers to a leaving group, e.g., a halogen (Hal) group).

A “second component piece” may have the following formula S2:

where P is an amino protection group suitable for use in peptidesynthesis, L₁ is hydroxyl or a carboxyl-activation group, and R₄ is asdefined above. Preferred protection groups include t-butyl dimethylsilyl(TBDMS), t-butyloxycarbonyl (BOC), methyloxycarbonyl (MOC),9H-fluorenylmethyloxycarbonyl (FMOC), and allyloxycarbonyl (Alloc).N-Protected amino acids are commercially available; for example, FMOCamino acids are available from a variety of sources. In order for thesecond component piece to be reactive with the first component piece, L₁is a carboxyl-activation group, and the conversion of carboxyl groups toactivated carboxyl groups may be readily achieved by methods known inthe art for the activation of carboxyl groups. Suitable activatedcarboxylic acid groups include acid halides where L₁ is a halide such aschloride or bromide, acid anhydrides where L₁ is an acyl group such asacetyl, reactive esters such as an N-hydroxysuccinimide esters andpentafluorophenyl esters, and other activated intermediates such as theactive intermediate formed in a coupling reaction using a carbodiimidesuch as dicyclohexylcarbodiimide (DCC). Accordingly, commerciallyavailable N-protected amino acids may be converted to carboxylicactivated forms by means-known to one of skill in the art.

In the case of the azido derivative of an amino acid serving as thesecond component piece, such compounds may be prepared from thecorresponding amino acid by the reaction disclosed by Zaloom et al. (J.Org. Chem. 46:5173-76, 1981).

Alternatively, the first component piece of the invention may have thefollowing formula S1′:

wherein R is as defined above and L₂ is a leaving group such as halogenatom or tosyl group, and the second component piece of the invention mayhave the following formula S2′:

wherein R₂, R₄ and P are as defined above,

A “third component piece” of this invention may have the followingformula S3:

where G, E, L₁ and L₂ are as defined above. Suitable third componentpieces are commercially available from a variety of sources or can beprepared by methods well known in organic chemistry.

In FIG. 1, the compound of formula (1) has —(C═O)— for A, —(CHR₄)— forB, —(C═O)— for D, and —(CR₆)— for E. Compounds of formula (1) wherein acarbonyl group is at position B and an R group is at position B, i.e.,compounds wherein A is —(CHR₃)— and B is —(C═O)—, may be prepared in amanner analogous to that shown in FIG. 1, as illustrated in FIG. 2. FIG.2 also illustrates adding a fourth component piece to thefirst-second-third component intermediate, rather than attaching thefourth component piece to the third component piece prior to reactionwith the first-second intermediate piece. In addition, FIG. 2illustrates the prepartion of compounds of the present invention whereinD is —(CHR₅)— (rather than —(C═O)— as in FIG. 1), and E is —(C═O)—(rather than —(CHR₆)— as in FIG. 1). Finally, FIG. 2 illustrates thepreparation of compounds wherein G is NR₇.

Thus, as illustrated above, the reverse-turn mimetic compounds offormula (I) may be synthesized by reacting a first component piece witha second component piece to yield a combined first-second intermediate,followed by reacting the combined first-second intermediate with thirdcomponent pieces sequentially to provide a combinedfirst-second-third-fourth intermediate, and then cyclizing thisintermediate to yield the reverse-turn mimetic structure.

The syntheses of representative component pieces of this invention aredescribed in Preparation Examples and working Examples.

The reverse-turn mimetic structures of formula (III) and (IV) may bemade by techniques analogous to the modular component synthesisdisclosed above, but with appropriate modifications to the componentpieces.

The reverse-turn mimetic structures of the present invention are usefulas bioactive agents, such as diagnostic, prophylactic, and therapeuticagents. For example, the reverse-turn mimetic structures of the presentinvention may be used for modulating a cell signaling transcriptionfactor related peptides in a warm-blooded animal, by a method comprisingadministering to the animal an effective amount of the compound offormula (I).

Further, the reverse-turn mimetic structures of the present inventionmay also be effective for inhibiting peptide binding to PTB domains in awarm-blooded animal; for modulating G protein coupled receptor (GPCR)and ion channel in a warm-blooded animal; for modulating cytokines in awarm-blooded animal.

Meanwhile, it has been found that the compounds of the formula (I),especially compounds of formula (VI) are effective for inhibiting ortreating disorders modulated by Wnt-signaling pathway, such as cancer,especially colorectal cancer.

wherein R_(a) is a phenyl group; a substituted phenyl group having oneor more substituents wherein the one or more substituents areindependently selected from one or more of amino, amidino, guanidino,hydrazino, amidazonyl, C₁₋₄alkylamino, C₁₋₄dialkylamino, halogen,perfluoro C₁₋₄alkyl, C₁₋₄alkyl, C₁₋₃alkoxy, nitro, carboxy, cyano,sulfuryl, and hydroxyl groups; a benzyl group; a substituted benzylgroup with one or more substituents where the one or more substituentsare independently selected from one or more of amino, amidino,guanidino, hydrazino, amidazonyl, C₁₋₄alkylamino, C₁₋₄dialkylamino,halogen, perfluoro C₁₋₄alkyl, C₁₋₃alkoxy, nitro, carboxy, cyano,sulfuryl, and hydroxyl group; or a bicyclic aryl group having 8 to 11ring members, which may have 1 to 3 heteroatoms selected from nitrogen,oxygen or sulfur; R_(b) is a monocyclic aryl group having 5 to 7 ringmembers, which may have 1 to 2 heteroatoms selected from nitrogen,oxygen or sulfur, and aryl ring in the compound may have one or moresubstituents selected from a group consisting of halide, hydroxy, cyano,lower alkyl, and lower alkoxy groups; Rc is a saturated or unsaturatedC₁₋₆alkyl, C₁₋₆alkoxy, perfluoro C₁₋₆alkyl group; and X₁, X₂, and X₃ maybe the same or different and independently selected from hydrogen,hydroxyl, and halide.

In another aspect, it is an object of the present invention to provide apharmaceutical composition comprising a safe and effective amount of thecompound having general formula (VI) and pharmaceutically acceptablecarrier, which can be used for treatment of disorders modulated by Wntsignaling pathway, especially by TCF4-β-catenin-CBP complex.

Further, the present invention is to provide a method for inhibiting thegrowth of tumor cells by using the above-described composition of thepresent invention; a method for inducing apoptosis of tumor cells byusing the above-described composition of the present invention; a methodfor treating a disorder modulated by TCF4-β catenin-CBP complex by usingthe above-described composition of the present invention; and a methodof treating cancer such as colorectal cancer by administering thecomposition of the present invention together with other anti-canceragent such as 5-fluorouracil (5-FU), taxol, cisplatin, mitomycin C,tegafur, raltitrexed, capecitabine, and irinotecan, etc.

In a preferred embodiment of the present invention, the compound of thepresent invention has a (6S,10R)-configuration as follows:

wherein R_(a) and R_(b) have the same meanings as defined above.

In another aspect of this invention, prodrugs derived from compoundshaving general formula (I) are disclosed. The prodrugs generallyincrease aqueous solubility and thus bioavailability of compounds havinggeneral formula (I). In certain embodiments, the prodrugs of the presentinvention have the following general formula (VII):(VI)—Y—R₁₀wherein (VI) is general formula (VI) as described above; Y is oxygen,sulfur, or nitrogen of a group selected from R_(a), R_(b), R_(c), X₁, X₂and X₃; R₁₀ is phosphate, hemisuccinate, hemimalate,phosphoryloxymethyloxycarbonyl, dimethylaminoacetate,dimethylaminoalkylcarbamate, hydroxyalkyl, amino acid, glycosyl,substituted or unsubstituted piperidine oxycarbonyl, or a salt thereof;and wherein the prodrugs are capable of serving as a substrate for aphosphatase or a carboxylase and are thereby converted to compoundshaving general formula (VI). In some embodiments, R₁₀ of the generalformula (VII) is not an amino acid group or a phospho-amino acid group.

In another aspect of this invention, libraries containing reverse-turnmimetic structures of the present invention are disclosed. Onceassembled, the libraries of the present invention may be screened toidentify individual members having bioactivity. Such screening of thelibraries for bioactive members may involve; for example, evaluating thebinding activity of the members of the library or evaluating the effectthe library members have on a functional assay. Screening is normallyaccomplished by contacting the library members (or a subset of librarymembers) with a target of interest, such as, for example, an antibody,enzyme, receptor or cell line. Library members which are capable ofinteracting with the target of interest, are referred to herein as“bioactive library members” or “bioactive mimetics”. For example, abioactive mimetic may be a library member which is capable of binding toan antibody or receptor, or which is capable of inhibiting an enzyme, orwhich is capable of eliciting or antagonizing a functional responseassociated, for example, with a cell line. In other words, the screeningof the libraries of the present invention determines which librarymembers are capable of interacting with one or more biological targetsof interest. Furthermore, when interaction does occur, the bioactivemimetic (or mimetics) may then be identified from the library members.The identification of a single (or limited number) of bioactivemimetic(s) from the library yields reverse-turn mimetic structures whichare themselves biologically active, and thus are useful as diagnostic,prophylactic or therapeutic agents, and may further be used tosignificantly advance identification of lead compounds in these fields.

Synthesis of the peptide mimetics of the library of the presentinvention may be accomplished using known peptide synthesis techniques,in combination with the first, second and third component pieces of thisinvention. More specifically, any amino acid sequence may be added tothe N-terminal and/or C-terminal of the conformationally constrainedreverse-turn mimetic. To this end, the mimetics may be synthesized on asolid support (such as PAM resin) by known techniques (see, e.g., JohnM. Stewart and Janis D. Young, Solid Phase Peptide Synthesis, 1984,Pierce Chemical Comp., Rockford, Ill.) or on a silyl-linked resin byalcohol attachment (see Randolph et al., J. Am Chem. Soc. 117:5712-14,1995).

In addition, a combination of both solution and solid phase synthesistechniques may be utilized to synthesize the peptide mimetics of thisinvention. For example, a solid support may be utilized to synthesizethe linear peptide sequence up to the point that the conformationallyconstrained reverse-turn is added to the sequence. A suitableconformationally constrained reverse-turn mimetic structure which hasbeen previously synthesized by solution synthesis techniques may then beadded as the next “amino acid” to the solid phase synthesis (i.e., theconformationally constrained reverse-turn mimetic, which has both anN-terminus and a C-terminus, may be utilized as the next amino acid tobe added to the linear peptide). Upon incorporation of theconformationally constrained reverse-turn mimetic structures into thesequence, additional amino acids may then be added to complete thepeptide bound to the solid support. Alternatively, the linear N-terminusand C-terminus protected peptide sequences may be synthesized on a solidsupport, removed from the support, and then coupled to theconformationally constrained reverse-turn mimetic structures in solutionusing known solution coupling techniques.

In another aspect of this invention, methods for constructing thelibraries are disclosed. Traditional combinatorial chemistry techniques(see, e.g., Gallop et al., J. Med. Chem. 37:1233-1251, 1994) permit avast number of compounds to be rapidly prepared by the sequentialcombination of reagents to a basic molecular scaffold. Combinatorialtechniques have been used to construct peptide libraries derived fromthe naturally occurring amino acids. For example, by taking 20 mixturesof 20 suitably protected and different amino acids and coupling eachwith one of the 20 amino acids, a library of 400 (i.e., 20²) dipeptidesis created. Repeating the procedure seven times results in thepreparation of a peptide library comprised of about 26 billion (i.e.,20⁸) octapeptides.

Specifically, synthesis of the peptide mimetics of the library of thepresent invention may be accomplished using known peptide synthesistechniques, for example, the General Scheme of [4,4,0] Reverse-TurnMimetic Library as follows:

Synthesis of the peptide mimetics of the libraries of the presentinvention was accomplished using a FlexChem Reactor Block which has 96well plates by known techniques. In the above scheme ‘Pol’ represents abromoacetal resin (Advanced ChemTech) and detailed procedure isillustrated below.

Step 1

A bromoacetal resin (37 mg, 0.98 mmol/g) and a solution of R₂-amine inDMSO (1.4 mL) were placed in a Robbins block (FlexChem) having 96 wellplates. The reaction mixture was shaken at 60° C. using a rotating oven[Robbins Scientific] for 12 hours. The resin was washed with DMF, MeOH,and then DCM

Step 2

A solution of commercial available FmocAmino Acids (4 equiv.), PyBob (4equiv:), HOAt (4 equiv.), and DIEA (12 equiv.) in DMF was added to theresin. After the reaction mixture was shaken for 12 hours at roomtemperature, the resin was washed with DMF, MeOH, and then DCM.

Step 3

To the resin swollen by DMF before reaction was added 25% piperidine inDMF and the reaction mixture was shaken for 30 min at room temperature.This deprotection step was repeated again and the resin was washed withDMF, Methanol, and then DCM. A solution of hydrazine acid (4 equiv.),HOBt (4 equiv.), and DIC (4 equiv.) in DMF was added to the resin andthe reaction mixture was shaken for 12 hours at room temperature. Theresin was washed with DMF, MeOH, and then DCM.

Step 4a (where hydrazine Acid is MOC Carbamate)

The resin obtained in Step 3 was treated with formic acid (1.2 mL eachwell) for 18 hours at room temperature. After the resin was removed byfiltration, the filtrate was condensed under a reduced pressure usingSpeedVac [SAVANT] to give the product as oil. The product was dilutedwith 50% water/acetonitrile and then lyophilized after freezing.

Step 4b (where Fmoc Hydrazine Acid is Used to Make Urea ThroughIsocynate)

To the resin swollen by DMF before reaction was added 25% piperidine inDMF and the reaction mixture was shaken for 30 min at room temperature.This deprotection step was repeated again and the resin was washed withDMF, Methanol, then DCM. To the resin swollen by DCM before reaction wasadded isocynate (5 equiv.) in DCM. After the reaction mixture was shakenfor 12 hours at room temperature the resin was washed with DMF, MeOH,then DCM. The resin was treated with formic acid (1.2 mL each well) for18 hours at room temperature. After the resin was removed by filtration,the filtrate was condensed under a reduced pressure using SpeedVac[SAVANT] to give the product as oil. The product was diluted with 50%water/acetonitrile and then lyophilized after freezing.

Step 4c (where Fmoc-Hydrazine Acid is Used to Make Urea Through ActiveCarbamate)

To the resin swollen by DMF before reaction was added 25% piperidine inDMF and the reaction mixture was shaken for 30 min at room temperature.This deprotection step was repeated again and the resin was washed withDMF, MeOH, and then DCM. To the resin swollen by DCM before reaction wasadded p-nitrophenyl chloroformate (5 equiv.) and diisopropyl ethylamine(5 equiv.) in DCM. After the reaction mixture was shaken for 12 hours atroom temperature, the resin was washed with DMF, MeOH, and then DCM. Tothe resin was added primary amines in DCM for 12 hours at roomtemperature and the resin was washed with DMF, MeOH, and then DCM. Afterreaction the resin was treated with formic acid (1.2 mL each well) for18 hours at room temperature. After the resin was removed by filtration,the filtrate was condensed under a reduced pressure using SpeedVac[SAVANT] to give the product as oil. The pro duct was diluted with 50%water/acetonitrile and then lyophilized after freezing.

To generate these block libraries the key intermediate hydrazine acidswere synthesized according to the procedure illustrated in PreparationExamples.

Tables 2A and 2B show a [4,4,0] Reverse turn mimetics library which canbe prepared according to the present invention, of which representativepreparation is given in Example 4.

TABLE 2A THE [4,4,0]REVERSE TURN MIMETICS LIBRARY

No R₂ R₄ R₇ R₁-Y′ Mol. Weight M + H 1 2,4-Cl_(2-benzyl) 4-HO-benzylAllyl OCH₃ 533 534 2 2,4-Cl_(2-benzyl) 4-NO₂-benzyl Allyl OCH₃ 562 563 32,4-Cl₂-benzyl 2,4-F₂-benzyl Allyl OCH₃ 553 554 4 2,4-Cl₂-benzyl4-Cl-benzyl Allyl OCH₃ 552 553 5 2,4-Cl₂-benzyl 2,2-bisphenylethyl AllylOCH₃ 594 595 6 2,4-Cl₂-benzyl 3-t-Bu-4-HO-benzy Allyl OCH₃ 590 591 72,4-Cl₂-benzyl 4-Me-benzyl Allyl OCH₃ 531 532 8 2,4-Cl₂-benzylCyclohexylmethyl Allyl OCH₃ 523 524 9 2,4-Cl₂-benzyl 4-F-benzyl AllylOCH₃ 535 536 10 2,4-Cl₂-benzyl 2-Cl-benzyl Allyl OCH₃ 552 553 112,4-Cl₂-benzyl 2,4-Cl₂-benzyl Allyl OCH₃ 586 587 12 2,4-Cl₂-benzylNaphth-2-ylmethyl Allyl OCH₃ 567 568 13 2,4-Cl₂-benzyl 4-HO-benzylBenzyl OCH₃ 583 584 14 2,4-Cl₂-benzyl 4-NO₂-benzyl Benzyl OCH₃ 612 61315 2,4-Cl₂-benzyl 2,4-F₂-benzyl Benzyl OCH₃ 603 604 16 2,4-Cl₂-benzyl4-Cl-benzyl Benzyl OCH₃ 602 603 17 2,4-Cl₂-benzyl 2,2-bisphenylethylBenzyl OCH₃ 644 645 18 2,4-Cl₂-benzyl 3-t-Bu-4-HO-benzy Benzyl OCH₃ 640641 19 2,4-Cl₂-benzyl 4-Me-benzyl Benzyl OCH₃ 582 583 20 2,4-Cl₂-benzylCyclohexylmethyl Benzyl OCH₃ 574 575 21 2,4-Cl₂-benzyl 4-F-benzyl BenzylOCH₃ 585 586 22 2,4-Cl₂-benzyl 2-Cl-benzyl Benzyl OCH₃ 602 603 232,4-Cl₂-benzyl 2,4-Cl₂-benzyl Benzyl OCH₃ 636 637 24 2,4-Cl₂-benzylNaphth-2-ylmethyl Benzyl OCH₃ 618 619 25 2,4-Cl₂-benzyl 4-HO-benzylAllyl OCH₃ 479 480 26 2,4-Cl₂-benzyl 4-NO₂-benzyl Allyl OCH₃ 508 509 272,4-Cl₂-benzyl 2,4-F₂-benzyl Allyl OCH₃ 499 500 28 2,4-Cl₂-benzyl4-Cl-benzyl Allyl OCH₃ 497 498 29 Phenethyl 2,2-bisphenylethyl AllylOCH₃ 539 540 30 Phenethyl 3-t-Bu-4-HO-benzy Allyl OCH₃ 535 536 31Phenethyl 4-Me-benzyl Allyl OCH₃ 477 478 32 Phenethyl CyclohexylmethylAllyl OCH₃ 469 470 33 Phenethyl 4-F-benzyl Allyl OCH₃ 481 482 34Phenethyl 2-Cl-benzyl Allyl OCH₃ 497 498 35 Phenethyl 2,4-Cl₂-benzylAllyl OCH₃ 531 532 36 Phenethyl Naphth-2-ylmethyl Allyl OCH₃ 513 514 37Phenethyl 4-HO-benzyl Benzyl OCH₃ 529 530 38 Phenethyl 4-NO₂-benzylBenzyl OCH₃ 558 559 39 Phenethyl 2,4-F₂-benzyl Benzyl OCH₃ 549 550 40Phenethyl 4-Cl-benzyl Benzyl OCH₃ 547 548 41 Phenethyl2,2-bisphenylethyl Benzyl OCH₃ 589 590 42 Phenethyl 3-t-Bu-4-HO-benzyBenzyl OCH₃ 585 586 43 Phenethyl 4-Me-benzyl Benzyl OCH₃ 527 528 44Phenethyl Cyclohexyl-methyl Benzyl OCH₃ 519 520 45 Phenethyl 4-F-benzylBenzyl OCH₃ 531 532 46 Phenethyl 2-Cl-benzyl Benzyl OCH₃ 547 548 47Phenethyl 2,4-Cl₂-benzyl Benzyl OCH₃ 582 583 48 PhenethylNaphth-2-ylmethyl Benzyl OCH₃ 563 564 49 Phenethyl 4-HO-benzyl AllylOCH₃ 497 498 50 Phenethyl 4-NO₂-benzyl Allyl OCH₃ 526 527 51 Phenethyl2,4-F₂-benzyl Allyl OCH₃ 517 518 52 Phenethyl 4-Cl-benzyl Allyl OCH₃ 515516 53 4-F-phenylethyl 2,2-bisphenylethyl Allyl OCH₃ 557 558 544-F-phenylethyl 3-t-Bu-4-HO-benzyl Allyl OCH₃ 553 554 55 4-F-phenylethyl4-Me-benzyl Allyl OCH₃ 495 496 56 4-F-phenylethyl Cyclohexyl-methylAllyl OCH₃ 487 488 57 4-F-phenylethyl 4-F-benzyl Allyl OCH₃ 499 500 584-F-phenylethyl 2-Cl-benzyl Allyl OCH₃ 515 516 59 4-F-phenylethyl2,4-Cl₂-benzyl Allyl OCH₃ 549 550 60 4-F-phenylethyl Naphth-2-ylmethylAllyl OCH₃ 531 532 61 4-F-phenylethyl 4-HO-benzyl Benzyl OCH₃ 547 548 624-F-phenylethyl 4-NO₂-benzyl Benzyl OCH₃ 576 577 63 4-F-phenylethyl2,4-F₂-benzyl Benzyl OCH₃ 567 568 64 4-F-phenylethyl 4-Cl-benzyl BenzylOCH₃ 565 566 65 4-F-phenylethyl 2,2-bisphenylethyl Benzyl OCH₃ 607 60866 4-F-phenylethyl 3-t-Bu-4-HO-benzyl Benzyl OCH₃ 603 604 674-F-phenylethyl 4-Me-benzyl Benzyl OCH₃ 545 546 68 4-F-phenylethylCyclohexyl-methyl Benzyl OCH₃ 537 538 69 4-F-phenylethyl 4-F-benzylBenzyl OCH₃ 549 550 70 4-F-phenylethyl 2-Cl-benzyl Benzyl OCH₃ 565 56671 4-F-phenylethyl 2,4-Cl₂-benzyl Benzyl OCH₃ 599 600 72 4-F-phenylethylNaphth-2-ylmethyl Benzyl OCH₃ 581 582 73 4-F-phenylethyl 4-HO-benzylAllyl OCH₃ 509 510 74 4-F-phenylethyl 4-NO₂-benzyl Allyl OCH₃ 538 539 754-F-phenylethyl 2,4-F₂-benzyl Allyl OCH₃ 529 530 76 4-F-phenylethyl4-Cl-benzyl Allyl OCH₃ 527 528 77 4-MeO-phenylethyl 2,2-bisphenylethylAllyl OCH₃ 569 570 78 4-MeO-phenylethyl 3-t-Bu-4-HO-benzyl Allyl OCH₃565 566 79 4-MeO-phenylethyl 4-Me-benzyl Allyl OCH₃ 507 508 804-MeO-phenylethyl Cyclohexyl-methyl Allyl OCH₃ 499 500 824-MeO-phenylethyl 2-Cl-benzyl Allyl OCH₃ 527 528 83 4-MeO-phenylethyl2,4-Cl₂-benzyl Allyl OCH₃ 561 562 84 4-MeO-phenylethyl Naphth-2-ylmethylAllyl OCH₃ 543 544 85 4-MeO-phenylethyl 4-HO-benzyl Benzyl OCH₃ 559 56086 4-MeO-phenylethyl 4-NO₂-benzyl Benzyl OCH₃ 588 589 874-MeO-phenylethyl 2,4-F₂-benzyl Benzyl OCH₃ 579 580 88 4-MeO-phenylethyl4-Cl-benzyl Benzyl OCH₃ 577 578 89 4-MeO-phenylethyl 2,2-bisphenylethylBenzyl OCH₃ 619 620 90 4-MeO-phenylethyl 3-t-Bu-4-HO-benzyl Benzyl OCH₃615 616 91 4-MeO-phenylethyl 4-Me-benzyl Benzyl OCH₃ 557 558 924-MeO-phenylethyl Cyclohexylmethyl Benzyl OCH₃ 549 550 934-MeO-phenylethyl 4-F-benzyl Benzyl OCH₃ 561 562 94 4-MeO-phenylethyl2-Cl-benzyl Benzyl OCH₃ 577 578 95 4-MeO-phenylethyl 2,4-Cl₂-benzylBenzyl OCH₃ 612 613 96 4-MeO-phenylethyl Naphth-2-ylmethyl Benzyl OCH₃593 594 97 Isoamyl 4-HO-benzyl Styrylmethyl OCH₃ 521 522 98 Isoamyl4-NO₂-benzyl Styrylmethyl OCH₃ 550 551 99 Isoamyl 2,4-F₂-benzylStyrylmethyl OCH₃ 541 542 100 Isoamyl 4-Cl-benzyl Styrylmethyl OCH₃ 539540 101 Isoamyl 2,2-bisphenylethyl Styrylmethyl OCH₃ 581 582 102 Isoamyl3-t-Bu-4-HO-benzyl Styrylmethyl OCH₃ 497 498 103 Isoamyl 4-Me-benzylStyrylmethyl OCH₃ 519 520 104 Isoamyl Cyclohexylmethyl Styrylmethyl OCH₃511 512 105 Isoamyl 4-F-benzyl Styrylmethyl OCH₃ 523 524 106 Isoamyl2-Cl-benzyl Styrylmethyl OCH₃ 539 540 107 Isoamyl 2,4-Cl₂-benzylStyrylmethyl OCH₃ 574 575 108 Isoamyl Naphth-2-ylmethyl StyrylmethylOCH₃ 555 556 109 Isoamyl 4-HO-benzyl 2,6-Cl₂-benzyl OCH₃ 563 564 110Isoamyl 4-NO2-benzyl 2,6-Cl₂-benzyl OCH₃ 592 593 111 Isoamyl2,4-F₂-benzyl 2,6-Cl₂-benzyl OCH₃ 583 584 112 Isoamyl 4-Cl-benzyl2,6-Cl₂-benzyl OCH₃ 582 583 113 Isoamyl 2,2-bisphenylethyl2,6-Cl₂-benzyl OCH₃ 624 625 114 isoamyl 3-t-Bu-4-HO-benzyl2,6-Cl₂-benzyl OCH₃ 540 541 115 Isoamyl 4-Me-benzyl 2,6-Cl₂-benzyl OCH₃562 563 116 Isoamyl Cyclohexylmethyl 2,6-Cl₂-benzyl OCH₃ 554 555 117Isoamyl 4-F-benzyl 2,6-Cl₂-benzyl OCH₃ 565 566 118 Isoamyl 2-Cl-benzyl2,6-Cl₂-benzyl OCH₃ 582 583 119 Isoamyl 2,4-Cl₂-benzyl 2,6-Cl₂-benzylOCH₃ 616 617 120 Isoamyl Naphth-2-ylmethyl 2,6-Cl₂-benzyl OCH₃ 598 599121 3-MeO-propyl 4-HO-benzyl Styrylmethyl OCH₃ 523 524 122 3-MeO-propyl4-NO_(2-benzyl) Styrylmethyl OCH₃ 552 553 123 3-MeO-propyl 2,4-F₂-benzylStyrylmethyl OCH₃ 543 544 124 3-MeO-propyl 4-Cl-benzyl Styrylmethyl OCH₃541 542 125 3-MeO-propyl 2,2-bisphenylethyl Styrylmethyl OCH₃ 583 584126 3-MeO-propyl 3-t-Bu-4-HO-benzyl Styrylmethyl OCH₃ 499 500 1273-MeO-propyl 4-Me-benzyl Styrylmethyl OCH₃ 521 522 128 3-MeO-propylCyclohexyl-methyl Styrylmethyl OCH₃ 513 514 129 3-MeO-propyl 4-F-benzylStyrylmethyl OCH₃ 525 526 130 3-MeO-propyl 2-Cl-benzyl Styrylmethyl OCH₃541 542 131 3-MeO-propyl 2,4-Cl₂-benzyl Styrylmethyl OCH₃ 575 576 1323-MeO-propyl Naphth-2-ylmethyl Styrylmethyl OCH₃ 557 558 1333-MeO-propyl 4-HO-benzyl 2,6-Cl₂-benzyl OCH₃ 565 566 134 3-MeO-propyl4-NO₂-benzyl 2,6-Cl₂-benzyl OCH₃ 594 595 135 3-MeO-propyl 2,4-F₂-benzyl2,6-Cl₂-benzyl OCH₃ 585 86 136 3-MeO-propyl 4-Cl-benzyl 2,6-Cl₂-benzylOCH₃ 584 585 137 3-MeO-propyl 2,2-bisphenylethyl 2,6-Cl₂-benzyl OCH₃ 626627 138 3-MeO-propyl 3-t-Bu-4-HO-benzyl 2,6-Cl₂-benzyl OCH₃ 541 542 1393-MeO-propyl 4-Me-benzyl 2,6-Cl₂-benzyl OCH₃ 563 564 140 3-MeO-propylCyclohexyl-methyl 2,6-Cl₂-benzyl OCH₃ 556 557 141 3-MeO-propyl4-F-benzyl 2,6-Cl₂-benzyl OCH₃ 567 568 142 3-MeO-propyl 2-Cl-benzyl2,6-Cl₂-benzyl OCH₃ 584 585 143 3-MeO-propyl 2,4-Cl₂-benzyl2,6-Cl₂-benzyl OCH₃ 618 619 144 3-MeO-propyl Naphth-2-ylmethyl2,6-Cl₂-benzyl OCH₃ 600 601 145 4-MeO-phenylethyl 4-HO-benzylStyrylmethyl OCH₃ 585 586 146 4-MeO-phenylethyl 4-NO2-benzylStyrylmethyl OCH₃ 614 615 147 4-MeO-phenylethyl 2,4-F₂-benzylStyrylmethyl OCH₃ 605 606 148 4-MeO-phenylethyl 4-Cl-benzyl StyrylmethylOCH₃ 603 604 149 4-MeO-phenylethyl 2,2-bisphenylethyl Styrylmethyl OCH₃645 646 150 4-MeO-phenylethyl 3-t-Bu-4-HO-benzyl Styrylmethyl OCH₃ 561562 151 4-MeO-phenylethyl 4-Me-benzyl Styrylmethyl OCH₃ 583 584 1524-MeO-phenylethyl Cyclohexyl-methyl Styrylmethyl OCH₃ 575 576 1534-MeO-phenylethyl 4-F-benzyl Styrylmethyl OCH₃ 587 588 1544-MeO-phenylethyl 2-Cl-benzyl Styrylmethyl OCH₃ 603 604 1554-MeO-phenylethyl 2,4-Cl₂-benzyl Styrylmethyl OCH₃ 638 639 1564-MeO-phenylethyl Naphth-2-ylmethyl Styrylmethyl OCH₃ 619 620 1574-MeO-phenylethyl 4-HO-benzyl 2,6-Cl₂-benzyl OCH₃ 628 629 1584-MeO-phenylethyl 4-NO₂-benzyl 2,6-Cl₂-benzyl OCH₃ 657 658 1594-MeO-phenylethyl 2,4-F₂-benzyl 2,6-Cl₂-benzyl OCH₃ 648 649 1604-MeO-phenylethyl 4-Cl-benzyl 2,6-Cl₂-benzyl OCH₃ 646 647 1614-MeO-phenylethyl 2,2-bisphenylethyl 2,6-Cl₂-benzyl OCH₃ 688 689 1624-MeO-phenylethyl 3-t-Bu-4-HO-benzyl 2,6-Cl₂-benzyl OCH₃ 604 605 1634-MeO-phenylethyl 4-Me-benzyl 2,6-Cl₂-benzyl OCH₃ 626 627 1644-MeO-phenylethyl Cyclohexylmethyl 2,6-Cl₂-benzyl OCH₃ 618 619 1654-MeO-phenylethyl 4-F-benzyl 2,6-Cl₂-benzyl OCH₃ 630 631 1664-MeO-phenylethyl 2-Cl-benzyl 2,6-Cl₂-benzyl OCH₃ 646 647 1674-MeO-phenylethyl 2,4-Cl₂-benzyl 2,6-Cl₂-benzyl OCH₃ 680 681 1684-MeO-phenylethyl Naphth-2-ylmethyl 2,6-Cl₂-benzyl OCH₃ 662 663 169Tetrahydrofuran-2- 4-HO-benzyl Styrylmethyl OCH₃ 535 536 ylmethyl 170Tetrahydrofuran-2- 4-NO₂-benzyl Styrylmethy OCH₃ 564 565 ylmethyl 171Tetrahydrofuran-2- 2,4-F_(2-benzyl) Styrylmethyl OCH₃ 555 556 ylmethyl172 Tetrahydrofuran-2- 4-Cl-benzyl Styrylmethyl OCH₃ 553 554 ylmethyl173 Tetrahydrofuran-2- 2,2-bisphenylethyl Styrylmethyl OCH₃ 595 596ylmethyl 174 Tetrahydrofuran-2- 3-t-Bu-4-HO-benzyl Styrylmethyl OCH₃ 511512 ylmethyl 175 Tetrahydrofuran-2- 4-Me-benzyl Styrylmethyl OCH₃ 533534 ylmethyl 176 Tetrahydrofuran-2- Cyclohexyl-methyl Styrylmethyl OCH₃525 526 ylmethyl 177 Tetrahydrofuran-2- 4-F-benzyl Styrylmethyl OCH₃ 537538 ylmethyl 178 Tetrahydrofuran-2- 2-Cl-benzyl Styrylmethyl OCH₃ 553554 ylmethyl 179 Tetrahydrofuran-2- 2,4-Cl₂-benzyl Styrylmethyl OCH₃ 588589 ylmethyl 180 Tetrahydrofuran-2- Naphth-2-ylmethyl Styrylmethyl OCH₃569 570 ylmethyl 181 Tetrahydrofuran-2- 4-HO-benzyl 2,6-Cl₂-benzyl OCH₃577 578 ylmethyl 182 Tetrahydrofuran-2- 4-NO₂-benzyl 2,6-Cl₂-benzyl OCH₃606 607 ylmethyl 183 Tetrahydrofuran-2- 2,4-F_(2-benzyl) 2,6-Cl₂-benzylOCH₃ 597 598 ylmethyl 184 Tetrahydrofuran-2- 4-Cl-benzyl 2,6-Cl₂-benzylOCH₃ 596 597 ylmethyl 185 Tetrahydrofuran-2- 2,2-bisphenylethyl2,6-Cl₂-benzyl OCH₃ 638 639 ylmethyl 186 Tetrahydrofuran-2-3-t-Bu-4-HO-benzyl 2,6-Cl₂-benzyl OCH₃ 553 554 ylmethyl 187Tetrahydrofuran-2- 4-Me-benzyl 2,6-Cl₂-benzyl OCH₃ 575 576 ylmethyl 188Tetrahydrofuran-2- Cyclohexyl-methyl 2,6-Cl₂-benzyl OCH₃ 568 569ylmethyl 189 Tetrahydrofuran-2- 4-F-benzyl 2,6-Cl₂-benzyl OCH₃ 579 580ylmethyl 190 Tetrahydrofuran-2- 2-Cl-benzyl 2,6-Cl₂-benzyl OCH₃ 596 597ylmethyl 191 Tetrahydrofuran-2- 2,4-Cl₂-benzyl 2,6-Cl₂-benzyl OCH₃ 630631 ylmethyl 192 Tetrahydrofuran-2- Naphth-2-ylmethyl 2,6-Cl₂-benzylOCH₃ 612 613 ylmethyl 193 Phenethyl 4-HO-benzyl Methyl(4-Me-phenyl)amino 528 529 194 Phenethyl 4-HO-benzyl Methyl(4-Cl-phenyl)amino 548 549 195 Phenethyl 4-HO-benzyl Methyl Phenylamino514 515 196 Phenethyl 4-HO-benzyl Methyl ((R)-α- 542 543methylbenzyl)amino 197 Phenethyl 4-HO-benzyl Methyl Benzylamino 528 529198 Phenethyl 4-HO-benzyl Methyl (4-MeO-phenyl)amino 544 545 199Phenethyl 4-HO-benzyl Methyl (4-Br-phenyl)amino 592 593 200 Phenethyl4-HO-benzyl Methyl (4-CF₃-phenyl)amino 582 583 201 Phenethyl 4-HO-benzylMethyl Pentylamino 508 509 202 Phenethyl 4-HO-benzyl Methyl(2-Phenylethyl)amino 542 543 203 Phenethyl 4-HO-benzyl Methyl(4-MeO-benzyl)amino 558 559 204 Phenethyl 4-HO-benzyl MethylCyclohexylamino 520 521 205 2,2-bisphenylethyl 4-HO-benzyl Methyl(4-Me-phenyl)amino 604 605 206 2,2-bisphenylethyl 4-HO-benzyl Methyl(4-Cl-phenyl)amino 624 625 207 2,2-bisphenylethyl 4-HO-benzyl MethylPhenylamino 590 591 208 2,2-bisphenylethyl 4-HO-benzyl Methyl ((R)-α-618 619 methylbenzyl)amino 209 2,2-bisphenylethyl 4-HO-benzyl MethylBenzylamino 604 605 210 2,2-bisphenylethyl 4-HO-benzyl Methyl(4-MeO-phenyl)amino 620 621 211 2,2-bisphenylethyl 4-HO-benzyl Methyl(4-Br-phenyl)amino 669 670 212 2,2-bisphenylethyl 4-HO-benzyl Methyl(4-CF₃-phenyl)amino 658 659 213 2,2-bisphenylethyl 4-HO-benzyl MethylPentylamino 584 585 214 2,2-bisphenylethyl 4-HO-benzyl Methyl(2-Phenylethyl)amino 618 619 215 2,2-bisphenylethyl 4-HO-benzyl Methyl(4-MeO-benzyl)amino 634 635 216 2,2-bisphenylethyl 4-HO-benzyl MethylCyclohexylamino 596 597 217 Phenethyl 3,4-Cl₂-benzyl Methyl(4-Me-phenyl)amino 581 582 218 Phenethyl 3,4-Cl₂-benzyl Methyl(4-Cl-phenyl)amino 601 602 219 Phenethyl 3,4-Cl₂-benzyl MethylPhenylamino 566 567 220 Phenethyl 3,4-Cl₂-benzyl Methyl ((R)-α- 595 596methylbenzyl)amino 221 Phenethyl 3,4-Cl₂-benzyl Methyl Benzylamino 581582 222 Phenethyl 3,4-Cl₂-benzyl Methyl (4-MeO-phenyl)amino 597 598 223Phenethyl 3,4-Cl₂-benzyl Methyl (4-Br-phenyl)amino 645 646 224 Phenethyl3,4-Cl₂-benzyl Methyl (4-CF₃-phenyl)amino 634 635 225 Phenethyl3,4-Cl₂-benzyl Methyl Pentylamino 561 562 226 Phenethyl 3,4-Cl₂-benzylMethyl (2-Phenylethyl)amino 595 596 227 Phenethyl 3,4-Cl₂-benzyl Methyl(4-MeO-benzyl)amino 611 612 228 Phenethyl 3,4-Cl₂-benzyl MethylCyclohexylamino 573 574 229 2,2-bisphenylethyl 3,4-Cl₂-benzyl Methyl(4-Me-phenyl)amino 657 658 230 2,2-bisphenylethyl 3,4-Cl₂-benzyl Methyl(4-Cl-phenyl)amino 677 678 231 2,2-bisphenylethyl 3,4-Cl₂-benzyl MethylPhenylamino 643 644 232 2,2-bisphenylethyl 3,4-Cl₂-benzyl Methyl ((R)-α-671 672 methylbenzyl)amino 233 2,2-bisphenylethyl 3,4-Cl₂-benzyl MethylBenzylamino 657 658 234 2,2-bisphenylethyl 3,4-Cl₂-benzyl Methyl(4-MeO-phenyl)amino 673 674 235 2,2-bisphenylethyl 3,4-Cl₂-benzyl Methyl(4-Br-phenyl)amino 721 722 236 2,2-bisphenylethyl 3,4-Cl₂-benzyl Methyl(4-CF₃-phenyl)amino 711 712 237 2,2-bisphenylethyl 3,4-Cl₂-benzyl MethylPentylamino 637 638 238 2,2-bisphenylethyl 3,4-Cl₂-benzyl Methyl(2-Phenylethyl)amino 671 672 239 2,2-bisphenylethyl 3,4-Cl₂-benzylMethyl (4-MeO-benzyl)amino 687 688 240 2,2-bisphenylethyl 3,4-Cl₂-benzylMethyl Cyclohexylamino 649 650 241 Isoamyl 4-HO-benzyl Methyl(4-Me-phenyl)amino 478 479 242 Isoamyl 4-HO-benzyl Methyl(4-Cl-phenyl)amino 498 499 243 Isoamyl 4-HO-benzyl Methyl Phenylamino464 465 244 Isoamyl 4-HO-benzyl Methyl ((R)-α- 492 493methylbenzyl)amino 245 Isoamyl 4-HO-benzyl Methyl Benzylamino 478 479246 Isoamyl 4-HO-benzyl Methyl (4-MeO-phenyl)amino 494 495 247 Isoamyl4-HO-benzyl Methyl (4-Br-phenyl)amino 542 543 248 Isoamyl 4-HO-benzylMethyl (4-CF₃-phenyl)amino 532 533 249 Isoamyl 4-HO-benzyl MethylPentylamino 458 459 250 Isoamyl 4-HO-benzyl Methyl (2-Phenylethyl)amino492 493 251 Isoamyl 4-HO-benzyl Methyl (4-MeO-benzyl)amino 508 509 252Isoamyl 4-HO-benzyl Methyl Cyclohexylamino 470 471 253 Isoamyl4-HO-benzyl Methyl (4-Me-phenyl)amino 554 555 254 Isoamyl 4-HO-benzylMethyl (4-Cl-phenyl)amino 574 575 255 Isoamyl 4-HO-benzyl MethylPhenylamino 540 541 256 Isoamyl 4-HO-benzyl Methyl ((R)-α- 568 569methylbenzyl)amino 257 Isoamyl 4-HO-benzyl Methyl Benzylamino 554 555258 Isoamyl 4-HO-benzyl Methyl (4-MeO-phenyl)amino 570 571 259 Isoamyl4-HO-benzyl Methyl (4-Br-phenyl)amino 619 620 260 Isoamyl 4-HO-benzylMethyl (4-CF₃-phenyl)amino 608 609 261 Isoamyl 4-HO-benzyl MethylPentylamino 534 535 262 Isoamyl 4-HO-benzyl Methyl (2-Phenylethyl)amino568 569 263 Isoamyl 4-HO-benzyl Methyl (4-MeO-benzyl)amino 584 585 264Isoamyl 4-HO-benzyl Methyl Cyclohexylamino 546 547 265 4-methylbenzyl3,4-Cl₂-benzyl Methyl (4-Me-phenyl)amino 526 527 266 4-methylbenzyl3,4-Cl₂-benzyl Methyl (4-Cl-phenyl)amino 546 547 267 4-methylbenzyl3,4-Cl₂-benzyl Methyl Phenylamino 512 513 268 4-methylbenzyl3,4-Cl₂-benzyl Methyl ((R)-α- 540 541 methylbenzyl)amino 2694-methylbenzyl 3,4-Cl₂-benzyl Methyl Benzylamino 526 527 2704-methylbenzyl 3,4-Cl₂-benzyl Methyl (4-MeO-phenyl)amino 542 543 2714-methylbenzyl 3,4-Cl₂-benzyl Methyl (4-Br-phenyl)amino 591 592 2724-methylbenzyl 3,4-Cl₂-benzyl Methyl (4-CF₃-phenyl)amino 580 581 2734-methylbenzyl 3,4-Cl₂-benzyl Methyl Pentylamino 506 507 2744-methylbenzyl 3,4-Cl₂-benzyl Methyl (2-Phenylethyl)amino 540 541 2754-methylbenzyl 3,4-Cl₂-benzyl Methyl (4-MeO-benzyl)amino 556 557 2764-methylbenzyl 3,4-Cl₂-benzyl Methyl Cyclohexylamino 518 519 2774-methylbenzyl 3,4-Cl₂-benzyl Methyl (4-Me-phenyl)amino 602 603 2784-methylbenzyl 3,4-Cl₂-benzyl Methyl (4-Cl-phenyl)amino 622 623 2794-methylbenzyl 3,4-Cl₂-benzyl Methyl Phenylamino 588 589 2804-methylbenzyl 3,4-Cl₂-benzyl Methyl ((R)-60 - 616 617methylbenzyl)amino 281 4-methylbenzyl 3,4-Cl₂-benzyl Methyl Benzylamino602 603 282 4-methylbenzyl 3,4-Cl₂-benzyl Methyl (4-MeO-phenyl)amino 618619 283 4-methylbenzyl 3,4-Cl₂-benzyl Methyl (4-Br-phenyl)amino 667 668284 4-methylbenzyl 3,4-Cl₂-benzyl Methyl (4-CF₃-phenyl)amino 656 657 2854-methylbenzyl 3,4-Cl₂-benzyl Methyl Pentylamino 582 583 2864-methylbenzyl 3,4-Cl₂-benzyl Methyl (2-Phenylethyl)amino 616 617 2874-methylbenzyl 3,4-Cl₂-benzyl Methyl (4-MeO-benzyl)amino 632 633 2884-methylbenzyl 3,4-Cl₂-benzyl Methyl Cyclohexylamino 594 595 289Naphth-1-ylmethyl 4-HO-benzyl Methyl (N-Cbz-3- 751 752 Indoleethyl)amino290 Naphth-1-ylmethyl 4-HO-benzyl Methyl (Naphth-2- 614 615ylmethyl)amino 291 Naphth-1-ylmethyl 4-HO-benzyl Methyl(2-Phenylethyl)amino 578 579 292 Naphth-1-ylmethyl 4-HO-benzyl Methyl(2-(4-MeO- 608 609 phenyl)ethyl]amino 293 Naphth-1-ylmethyl 4-HO-benzylMethyl (3-CF₃-benzyl)amino 632 633 294 Naphth-1-ylmethyl 4-HO-benzylMethyl (4-MeO-benzyl)amino 594 595 295 Naphth-1-ylmethyl 4-HO-benzylMethyl (4-F-phenylethyl)-amino 596 597 296 Naphth-1-ylmethyl 4-HO-benzylMethyl (3,4-Cl₂-benzyl)amino 633 634 297 Naphth-1-ylmethyl 4-HO-benzylMethyl (2-HO-ethyl)amino 518 519 298 Naphth-1-ylmethyl 4-HO-benzylMethyl (3-MeO-propyl)amino 546 547 299 Naphth-1-ylmethyl 4-HO-benzylMethyl (Tetrahydrofuran-2- 558 559 ylmethyl)amino 300 Naphth-1-ylmethyl4-HO-benzyl Methyl (cyclohexylmethyl)amio 570 571 301 Naphth-1-ylmethyl4-HO-benzyl Propyl (N-Cbz-3- 779 780 Indoleethyl)amino 302Naphth-1-ylmethyl 4-HO-benzyl Propyl (Naphth-2- 642 643 ylmethyl)amino303 Naphth-1-ylmethyl 4-HO-benzyl Propyl (2-Phenylethyl)amino 606 607304 Naphth-1-ylmethyl 4-HO-benzyl Propyl [2-(4-MeO- 636 637phenyl)ethyl]amino 305 Naphth-1-ylmethyl 4-HO-benzyl Propyl(3-CF₃-benzyl)amino 660 661 306 Naphth-1-ylmethyl 4-HO-benzyl Propyl(4-MeO-benzyl)amino 622 623 307 Naphth-1-ylmethyl 4-HO-benzyl Propyl(4-F-phenylethyl)amino 624 625 308 Naphth-1-ylmethyl 4-HO-benzyl Propyl(3,4-Cl₂-benzyl)amino 661 662 309 Naphth-1-ylmethyl 4-HO-benzyl Propyl(2-HO-ethyl)amino 546 547 310 Naphth-1-ylmethyl 4-HO-benzyl Propyl(3-MeO-propyl)amino 574 575 311 Naphth-1-ylmethyl 4-HO-benzyl Propyl(Tetrahydrofuran-2- 586 587 ylmethyl)amino 312 Naphth-1-ylmethyl4-HO-benzyl Propyl (cyclohexylmethyl)amino 598 599 313 Naphth-1-ylmethyl3,4-F₂-benzyl Methyl (N-Cbz-3- 771 772 Indoleethyl)amino 314Naphth-1-ylmethyl 3,4-F₂-benzyl Methyl (Naphth-2- 634 635 ylmethyl)amino315 Naphth-1-ylmethyl 3,4-F₂-benzyl Methyl (2-Phenylethyl)amino 598 599316 Naphth-1-ylmethyl 3,4-F₂-benzyl Methyl (2-(4-MeO- 628 629phenyl)ethyl]amino 317 Naphth-1-ylmethyl 3,4-F₂-benzyl Methyl(3-CF₃-benzyl)amino 652 653 318 Naphth-1-ylmethyl 3,4-F₂-benzyl Methyl(4-MeO-benzyl)amino 614 615 319 Naphth-1-ylmethyl 3,4-F₂-benzyl Methyl(4-F-phenylethyl)amino 616 617 320 Naphth-1-ylmethyl 3,4-F₂-benzylMethyl (3,4-Cl₂-benzyl)amino 653 654 321 Naphth-1-ylmethyl 3,4-F₂-benzylMethyl (2-HO-ethyl)amino 538 539 322 Naphth-1-ylmethyl 3,4-F₂-benzylMethyl (3-MeO-propyl)amino 566 567 323 Naphth-1-ylmethyl 3,4-F₂-benzylMethyl (Tetrahydrofuran-2- 578 579 ylmethyl)amino 324 Naphth-1-ylmethyl3,4-F₂-benzyl Methyl cyclohexylmethyl)amino 590 591 325Naphth-1-ylmethyl 3,4-F₂-benzyl Propyl (N-Cbz-3- 799 800Indoleethyl)amino 326 Naphth-1-ylmethyl 3,4-F₂-benzyl Propyl (Naphth-2-662 663 ylmethyl)amino 327 Naphth-1-ylmethyl 3,4-F₂-benzyl Propyl(2-Phenylethyl)amino 626 627 328 Naphth-1-ylmethyl 3,4-F₂-benzyl Propyl[2-(4-MeO- 656 657 phenyl)ethyl]amino 329 Naphth-1-ylmethyl3,4-F₂-benzyl Propyl (3-CF₃-benzyl)amino 680 681 330 Naphth-1-ylmethyl3,4-F₂-benzyl Propyl (4-MeO-benzyl)amino 642 643 331 Naphth-1-ylmethyl3,4-F₂-benzyl Propyl (4-F-phenylethyl)amino 644 645 332Naphth-1-ylmethyl 3,4-F₂-benzyl Propyl (3,4-Cl₂-benzyl)amino 681 682 333Naphth-1-ylmethyl 3,4-F₂-benzyl Propyl (2-HO-ethyl)amino 566 567 334Naphth-1-ylmethyl 3,4-F₂-benzyl Propyl (3-MeO-propyl)amino 594 595 335Naphth-1-ylmethyl 3,4-F₂-benzyl Propyl (Tetrahydrofuran-2- 606 607ylmethyl)amino 336 Naphth-1-ylmethyl 3,4-F₂-benzyl Propyl(cyclohexylmethyl)amino 618 619 337 Naphth-1-ylmethyl4-biphenylyl-methyl Methyl (N-Cbz-3- 811 812 Indoleethyl)amino 338Naphth-1-ylmethyl 4-biphenylylmethyl Methyl (Naphth-2- 674 675ylmethyl)amino 339 Naphth-1-ylmethyl 4-biphenylylmethyl Methyl(2-Phenylethyl)amino 638 639 340 Naphth-1-ylmethyl 4-biphenylylmethylMethyl [2-(4-MeO- 668 669 phenyl)ethyl]amino 341 Naphth-1-ylmethyl4-biphenylylmethyl Methyl (3-CF₃-benzyl)amino 692 693 342Naphth-1-ylmethyl 4-biphenylylmethyl Methyl (4-MeO-benzyl)amino 654 655343 Naphth-1-ylmethyl 4-biphenylylmethyl Methyl (4-F-phenylethyl)amino656 657 344 Naphth-1-ylmethyl 4-biphenylylmethyl Methyl(3,4-Cl₂-benzyl)amino 693 694 345 Naphth-1-ylmethyl 4-biphenylylmethylMethyl (2-HO-ethyl)amino 578 579 346 Naphth-1-ylmethyl4-biphenylylmethyl Methyl (3-MeO-propyl)amino 606 607 347Naphth-1-ylmethyl 4-biphenylylmethyl Methyl (Tetrahydrofuran-2- 618 619ylmethyl)amino 348 Naphth-1-ylmethyl 4-biphenylylmethyl Methyl(cyclohexylmethyl)amino 630 631 349 Naphth-1-ylmethyl 4-biphenylylmethylPropyl (N-Cbz-3- 839 840 Indoleethyl)amino 350 Naphth-1-ylmethyl4-biphenylylmethyl Propyl (Naphth-2- 702 703 ylmethyl)amino 351Naphth-1-ylmethyl 4-biphenylylmethyl Propyl (2-Phenylethyl)amino 666 667352 Naphth-1-ylmethyl 4-biphenylylmethyl Propyl [2-(4-MeO- 696 697phenyl)ethyl]amino 353 Naphth-1-ylmethyl 4-biphenylylmethyl Propyl(3-CF₃-benzyl)amino 720 721 354 Naphth-1-ylmethyl 4-biphenylylmethylPropyl (4-MeO-benzyl)amino 682 683 355 Naphth-1-ylmethyl4-biphenylylmethyl Propyl (4-F-phenylethyl)amino 684 685 356Naphth-1-ylmethyl 4-biphenylylmethyl Propyl (3,4-Cl₂-benzyl)amino 721722 357 Naphth-1-ylmethyl 4-biphenylylmethyl Propyl (2-HO-ethyl)amino606 607 358 Naphth-1-ylmethyl 4-biphenylylmethyl Propyl(3-MeO-propyl)amino 634 635 359 Naphth-1-ylmethyl 4-biphenylylmethylPropyl (Tetrahydrofuran-2- 646 647 ylmethyl)amino 360 Naphth-1-ylmethyl4-biphenylylmethyl Propyl (cyclohexylmethyl)amino 658 659 361Naphth-1-ylmethyl 3-t-Bu-4-HO-benzyl Methyl (N-Cbz-3- 807 808Indoleethyl)amino 362 Naphth-1-ylmethyl 3-t-Bu-4-HO-benzyl Methyl(Naphth-2- 670 671 ylmethyl)amino 363 Naphth-1-ylmethyl3-t-Bu-4-HO-benzyl Methyl (2-Phenylethyl)amino 634 635 364Naphth-1-ylmethyl 3-t-Bu-4-HO-benzyl Methyl [2-(4-MeO- 664 665phenyl)ethyl]amino 365 Naphth-1-ylmethyl 3-t-Bu-4-HO-benzyl Methyl(3-CF₃-benzyl)amino 688 689 366 Naphth-1-ylmethyl 3-t-Bu-4-HO-benzylMethyl (4-MeO-benzyl)amino 650 651 367 Naphth-1-ylmethyl3-t-Bu-4-HO-benzyl Methyl (4-F-phenylethyl)amino 652 653 368Naphth-1-ylmethyl 3-t-Bu-4-HO-benzyl Methyl (3,4-Cl₂-benzyl)amino 689690 369 Naphth-1-ylmethyl 3-t-Bu-4-HO-benzyl Methyl (2-HO-ethyl)amino574 575 370 Naphth-1-ylmethyl 3-t-Bu-4-HO-benzyl Methyl(3-MeO-propyl)amino 602 603 371 Naphth-1-ylmethyl 3-t-Bu-4-HO-benzylMethyl (Tetrahydrofuran-2- 614 615 ylmethyl)amino 372 Naphth-1-ylmethyl3-t-Bu-4-HO-benzyl Methyl (cyclohexylmethyl)amino 626 627 373Naphth-1-ylmethyl 3-t-Bu-4-HO-benzyl Propyl (N-Cbz-3- 835 836Indoleethyl)amino 374 Naphth-1-ylmethyl 3-t-Bu-4-HO-benzyl Propyl(Naphth-2- 698 699 ylmethyl)amino 375 Naphth-1-ylmethyl3-t-Bu-4-HO-benzyl Propyl (2-Phenylethyl)amino 662 663 376Naphth-1-ylmethyl 3-t-Bu-4-HO-benzyl Propyl [2-(4-MeO- 692 693phenyl)ethyl]amino 377 Naphth-1-ylmethyl 3-t-Bu-4-HO-benzyl Propyl(3-CF₃-benzyl)amino 716 717 378 Naphth-1-ylmethyl 3-t-Bu-4-HO-benzylPropyl (4-MeO-benzyl)amino 678 679 379 Naphth-1-ylmethyl3-t-Bu-4-HO-benzyl Propyl (4-F-phenylethyl)amino 680 681 380Naphth-1-ylmethyl 3-t-Bu-4-HO-benzyl Propyl (3,4-Cl₂-benzyl)amino 717718 381 Naphth-1-ylmethyl 3-t-Bu-4-HO-benzyl Propyl (2-HO-ethyl)amino602 603 382 Naphth-1-ylmethyl 3-t-Bu-4-HO-benzyl Propyl(3-MeO-propyl)amino 630 631 383 Naphth-1-ylmethyl 3-t-Bu-4-HO-benzylPropyl (Tetrahydrofuran-2- 642 643 ylmethyl)amino 384 Naphth-1-ylmethyl3-t-Bu-4-HO-benzyl Propyl (cyclohexylmethyl)amino 654 655 3854-Methoxybenzyl OCH₃ 5-F-benzyl OCH₃ 470 471 386 Naphthyl-1-ylmethyl4-HO-benzyl Styrylmethyl OCH₃ 591 592 387 Naphthyl-1-ylmethyl4-NO₂-benzyl Styrylmethyl OCH₃ 620 621 388 Naphthyl-1-ylmethyl3,4-F₂-benzyl Styrylmethyl OCH₃ 611 612 389 Naphthyl-1-ylmethyl4-Cl-benzyl Styrylmethyl OCH₃ 609 610 390 Naphthyl-1-ylmethyl4-Phenyl-benzyl Styrylmethyl OCH₃ 651 652 391 Naphthyl-1-ylmethyl3-t-Bu-4-HO-benzyl Styrylmethyl OCH₃ 647 648 392 Naphthyl-1-ylmethyl4-Methyl-benzyl Styrylmethyl OCH₃ 589 590 393 Naphthyl-1-ylmethylCyclohexylmethyl Styrylmethyl OCH₃ 581 582 394 Naphthyl-1-ylmethyl4-F-benzyl Styrylmethyl OCH₃ 593 594 395 Naphthyl-1-ylmethyl 2-Cl-benzylStyrylmethyl OCH₃ 609 610 396 Naphthyl-1-ylmethyl 3,4-Cl₂-benzylStyrylmethyl OCH₃ 644 645 397 Naphthyl-1-ylmethyl Naphthyl-1-ylmethylStyrylmethyl OCH₃ 625 626 398 3,4-Cl₂-benzyl 4-HO-benzyl StyrylmethylOCH₃ 610 611 399 3,4-Cl₂-benzyl 4-NO₂-benzyl Styrylmethyl OCH₃ 639 640400 3,4-Cl₂-benzyl 3,4-F₂-benzyl Styrylmethyl OCH₃ 629 630 4013,4-Cl₂-benzyl 4-Cl-benzyl Styrylmethyl OCH₃ 628 629 402 3,4-Cl₂-benzyl4-Phenyl-benzyl Styrylmethyl OCH₃ 670 671 403 3,4-Cl₂-benzyl3-t-Bu-4-HO-benzyl Styrylmethyl OCH₃ 666 667 404 3,4-Cl₂-benzyl4-Methyl-benzyl Styrylmethyl OCH₃ 608 609 405 3,4-Cl₂-benzylCyclohexylmethyl Styrylmethyl OCH₃ 600 601 406 3,4-Cl₂-benzyl 4-F-benzylStyrylmethyl OCH₃ 611 612 407 3,4-Cl₂-benzyl 2-Cl-benzyl StyrylmethylOCH₃ 628 629 408 3,4-Cl₂-benzyl 3,4-Cl₂-benzyl Styrylmethyl OCH₃ 662 663409 3,4-Cl₂-benzyl Naphthyl-1-ylmethyl Styrylmethyl OCH₃ 644 645 410Naphthyl-1-ylmethyl 4-HO-benzyl 2,6-Cl₂-benzyl OCH₃ 634 635 411Naphthyl-1-ylmethyl 4-NO₂-benzyl 2,6-Cl₂-benzyl OCH₃ 663 664 412Naphthyl-1-ylmethyl 3,4-F₂-benzyl 2,6-Cl₂-benzyl OCH₃ 654 655 413Naphthyl-1-ylmethyl 4-Cl-benzyl 2,6-Cl₂-benzyl OCH₃ 652 653 414Naphthyl-1-ylmethyl 4-Phenyl-benzyl 2,6-Cl₂-benzyl OCH₃ 694 695 415Naphthyl-1-ylmethyl 3-t-Bu-4-HO-benzyl 2,6-Cl₂-benzyl OCH₃ 690 691 416Naphthyl-1-ylmethyl 4-Methyl-benzyl 2,6-Cl₂-benzyl OCH₃ 632 633 417Naphthyl-1-ylmethyl Cyclohexylmethyl 2,6-Cl₂-benzyl OCH₃ 624 625 418Naphthyl-1-ylmethyl 4-F-benzyl 2,6-Cl₂-benzyl OCH₃ 636 637 419Naphthyl-1-ylmethyl 2-Cl-benzyl 2,6-Cl₂-benzyl OCH₃ 652 653 420Naphthyl-1-ylmethyl 3,4-Cl₂-benzyl 2,6-Cl₂-benzyl OCH₃ 686 687 421Naphthyl-1-ylmethyl Naphthyl-1-ylmethyl 2,6-Cl₂-benzyl OCH₃ 668 669 4223,4-Cl₂-benzyl 4-HO-benzyl 2,6-Cl₂-benzyl OCH₃ 652 653 4233,4-Cl₂-benzyl 4-NO₂-benzyl 2,6-Cl₂-benzyl OCH₃ 681 682 4243,4-Cl₂-benzyl 3,4-F₂-benzyl 2,6-Cl₂-benzyl OCH₃ 672 673 4253,4-Cl₂-benzyl 4-Cl-benzyl 2,6-Cl₂-benzyl OCH₃ 671 672 4263,4-Cl₂-benzyl 4-Phenyl-benzyl 2,6-Cl₂-benzyl OCH₃ 712 713 4273,4-Cl₂-benzyl 3-t-Bu-4-HO-benzyl 2,6-Cl₂-benzyl OCH₃ 708 709 4283,4-Cl₂-benzyl 4-Methyl-benzyl 2,6-Cl₂-benzyl OCH₃ 650 651 4293,4-Cl₂-benzyl Cyclohexylmethyl 2,6-Cl₂-benzyl OCH₃ 642 643 4303,4-Cl₂-benzyl 4-F-benzyl 2,6-Cl₂-benzyl OCH₃ 654 655 431 3,4-Cl₂-benzyl2-Cl-benzyl 2,6-Cl₂-benzyl OCH₃ 671 672 432 3,4-Cl₂-benzyl3,4-Cl₂-benzyl 2,6-Cl₂-benzyl OCH₃ 705 706 433 3,4-Cl₂-benzylNaphthyl-1-ylmethyl 2,6-Cl₂-benzyl OCH₃ 686 687 4342-Piperidin-1-yl-ethyl (S)-4-HO-benzyl Methyl Benzylamino 535 536 4352-Pipendin-1-yl- (S)-4-HO-benzyl Methyl ethylamino 604 6053,4-Cl₂-benzyl 436 3,4-Cl₂-benzyl (S)-4-HO-benzyl Methyl2-(1-Methyl-pyrrolidin- 604 605 2-yl)-ethylamino 437 3-Pyridylmethyl(S)-4-HO-benzyl Methyl 3,4-Cl₂-benzylamino 583 584 4382-Morpholin-4-yl-ethyl (S)-4-HO-benzyl Methyl 3,4-Cl₂-benzylamino 606607 439 3,4-Cl₂-benzyl (S)-4-HO-benzyl Methyl 3-Pyridylmethylamino 583584 440 3,4-Cl₂-benzyl (S)-4-HO-benzyl Methyl 2-Morpholin-4-yl- 606 607ethylamino 441 Naphthyl-1-ylmethyl 4-HO-benzyl Methyl 3-Imidazol-1-yl-582 583 propylamino 442 Naphthyl-1-ylmethyl 4-HO-benzyl Methyl 4- 593594 Aminophenethylamino 443 Naphthyl-1-ylmethyl 4-HO-benzyl Methyl3-Pyridylmethylamino 565 566 444 Naphthyl-1-ylmethyl 4-HO-benzyl Methyl2-(3-Pyridylethyl)amino 579 580 445 Naphthyl-1-ylmethyl 4-HO-benzylMethyl 4-Pyridylmethylamino 565 566 446 Naphthyl-1-ylmethyl 4-HO-benzylMethyl Benzyloxycarbonylamino 622 623 447 Naphthyl-1-ylmethyl4-HO-benzyl Methyl 4-F-benzylamino 582 583 448 Naphthyl-1-ylmethyl4-HO-benzyl Methyl 4-CO2H-benzylamino 608 609 449 Naphthyl-1-ylmethyl4-HO-benzyl Methyl 4-CF₃-benzylamino 632 633 450 Naphthyl-1-ylmethyl4-HO-benzyl Methyl (S)-alpha- 578 579 methylbenzylamino 451Naphthyl-1-ylmethyl 4-HO-benzyl Methyl (R)-alpha- 578 579methylbenzylamino 452 Naphthyl-1-ylmethyl 4-HO-benzyl Methyl2-F-benzylamino 582 583 453 Naphthyl-1-ylmethyl 4-HO-benzyl MethylDimethoxybenzylamino 624 625 454 Naphthyl-1-ylmethyl 4-HO-benzyl MethylCyanomethylamino 513 514 455 Naphthyl-1-ylmethyl 4-HO-benzyl MethylPhenylhydrazino 565 566 456 Naphthyl-1-ylmethyl 4-HO-benzyl Methyl4-Aminobenzylamino 579 580 457 Naphthyl-1-ylmethyl 4-HO-benzyl Methyl(S,S){2-[(2-hydroxy-1- 693 694 methyl-2-phenyl-ethyl)- methyl-carbamoyl]ethyl}-amino 458 Naphthyl-1-ylmethyl 4-HO-benzyl Methyl[4-(1,3-dioxo-1,3- 715 716 dihydro- isoindol-2-ylmethyl)- cyclohexyl]-methylamino 459 Naphthyl-1-ylmethyl 4-HO-benzyl Methyl Indan-1-ylamino590 591 460 Naphthyl-1-ylmethyl 4-HO-benzyl Methyl PhenylGlycine 622 623461 Naphthyl-1-ylmethyl 4-HO-benzyl Methyl 2,6-F₂-benzylamino 600 601462 Naphthyl-1-ylmethyl 4-HO-benzyl Methyl 3-F-benzylamino 582 583 463Naphthyl-1-ylmethyl 4-HO-benzyl Methyl Benzimidazol-2-yl- 604 605 amino464 Naphthyl-1-ylmethyl 4-HO-benzyl Methyl Diphenylmethylamino 640 641465 Naphthyl-1-ylmethyl 4-HO-benzyl Methyl Furan-2-yl-methylamino 554555 466 Naphthyl-1-ylmethyl 4-HO-benzyl Methyl 4-Dimethylamino- 607 608benzylamino 467 Naphthyl-1-ylmethyl 4-HO-benzyl Methyl Thiofuran-2-yl-584 585 methylamino 468 Naphthyl-1-ylmethyl 4-HO-benzyl Methyl4-NO₂-benzylamino 609 610 469 Naphthyl-1-ylmethyl 4-HO-benzyl Methyl BnO565 566 470 4-Methoxy-naphthyl- 4-HO-benzyl Methyl Benzylamino 594 5951-ylmethyl 471 Naphthyl-1-ylmethyl 4-HO-benzyl Methyl Phenethyl 563 564472 Naphthyl-1-ylmethyl 4-Methoxy-benzyl Methyl Benzylamino 578 579 473Naphthyl-1-ylmethyl 4-HO-benzyl Methyl 4-CF₃-phenylamino 618 619 474Naphthyl-1-ylmethyl 4-NO₂-benzyl Methyl 4-CF₃-phenylamino 647 648 475Naphthyl-1-ylmethyl 4-NO₂-benzyl Methyl Benzylamino 593 594 476 BenzylNaphthyl-1-ylmethyl 4-CN-benzyl OCH₃ 574 575 477 Thiofuran-2-yl-methylNaphthyl-1-ylmethyl 4-CN-benzyl OCH₃ 594 595 478 4-Dimethylamino-Naphthyl-1-ylmethyl 4-CN-benzyl OCH₃ 617 618 benzyl 479 PhenethylNaphthyl-1-ylmethyl 4-CN-benzyl OCH₃ 588 589 480 8-Quinoline-1yl-4-HO-benzyl Methyl Benzylamino 565 566 methyl 481 4-PyridylmethylNaphthyl-1-ylmethyl Benzyl OCH₃ 550 551 482 3.4-DimethoxybenzylNaphthyl-1-ylmethyl Benzyl OCH₃ 609 610 483 3,4-Dimethoxy-Naphthyl-1-ylmethyl Benzyl OCH₃ 623 624 phenethyl 484Thiofuran-2-yl-methyl Naphthyl-1-ylmethyl Benzyl OCH₃ 569 570 485Naphthyl-1-ylmethyl 3-Pyridylmethyl Methyl Benzylamino 549 550 486Naphthyl-1-ylmethyl Pentafluorobenzyl Methyl Benzylamino 638 639 487Naphthyl-1-ylmethyl 3-F-4-HO-benzyl Methyl Benzylamino 582 583 4884-F-phenethyl 4-Methyl-benzyl Methyl 4-CF₃-phenylamino 598 599 4894-Methoxyphenethyl 4-Methyl-benzyl Methyl 4-CF₃-phenylamino 610 611 4903,4-Dimethoxy- 4-Methyl-benzyl Methyl 4-CF₃-phenylamino 640 641phenethyl 491 Naphthyl-1-ylmethyl 4-Methyl-benzyl Methyl4-CF₃-phenylamino 616 617 492 3,4-Dimethoxybenzyl Naphthyl-1-ylmethyl4-CN-benzyl OCH₃ 634 635 493 3,4-Dimethoxy- Naphthyl-1-ylmethyl4-CN-benzyl OCH₃ 648 649 phenethyl 494 4-Quinoline-1yl- 4-HO-benzylMethyl Benzylamino 565 566 methyl 495 2-Pyridylmethyl 4-Methyl-benzylMethyl 4-CF₃-phenylamino 567 568 496 3-Pyridylmethyl 4-Methyl-benzylMethyl 4-CF₃-phenylamino 567 568 497 3,4-Dimethoxybenzyl 4-Methyl-benzylMethyl 4-CF₃-phenylamino 626 627 498 4-Methyl-benzyl 4-Methyl-benzylMethyl 4-CF₃-phenylamino 580 581 499 Thiofuran-2-yl-methyl4-Methyl-benzyl Methyl 4-CF₃-phenylamino 572 573 500 4-CF₃-benzyl4-Methyl-benzyl Methyl 4-CF₃-phenylamino 634 635 501 2,6-F₂-benzyl4-Methyl-benzyl Methyl 4-CF₃-phenylamino 602 603 502 4-F-benzyl4-Methyl-benzyl Methyl 4-CF₃-phenylamino 584 585 503Thiofuran-2-yl-ethyl 4-Methyl-benzyl Methyl 4-CF₃-phenylamino 586 587504 3,4-Cl₂-benzyl 4-Methyl-benzyl Methyl 4-CF₃-phenylamino 634 635 5054-CO₂H-Benzyl 4-HO-benzyl Methyl Benzylamino 558 559 506Naphthyl-1-ylmethyl 3-t-Bu-4-HO-benzyl Methyl Benzylamino 620 621 507Naphthyl-1-ylmethyl 3,4-(OH)2-benzyl Methyl Benzylamino 580 581 5082-F-benzyl 4-HO-benzyl Methyl Benzylamino 532 533 509 3-F-benzyl4-HO-benzyl Methyl Benzylamino 532 533 510 4-F-benzyl 4-HO-benzyl MethylBenzylamino 532 533 511 2,4-F₂-benzyl 4-HO-benzyl Methyl Benzylamino 550551 512 2,6-F₂-benzyl 4-HO-benzyl Methyl Benzylamino 550 551 5132,5-F₂-benzyl 4-HO-benzyl Methyl Benzylamino 550 551 514 3-CF₃-benyl4-HO-benzyl Methyl Benzylamino 582 583 515 4-CF₃-benyl 4-HO-benzylMethyl Benzylamino 582 583 516 3,4,5-F₃-benyl 4-HO-benzyl MethylBenzylamino 568 569 517 2-Cl-benzyl 4-HO-benzyl Methyl Benzylamino 548549 518 3-Cl-benzyl 4-HO-benzyl Methyl Benzylamino 548 549 5192,4-Cl₂-benzyl 4-HO-benzyl Methyl Benzylamino 582 583 520(S)-Methylphenyl 4-HO-benzyl Methyl Benzylamino 528 529 521(R)-Methylphenyl 4-HO-benzyl Methyl Benzylamino 528 529 5224-Methyl-benzyl 4-HO-benzyl Methyl Benzylamino 528 529 5234-Methoxybenzyl 4-HO-benzyl Methyl Benzylamino 544 545 5243,4-Dimethoxybenzyl 4-HO-benzyl Methyl Benzylamino 574 575 525Furan-2-yl- 4-HO-benzyl Methyl Benzylamino 504 505 methylamino 526(R)-Methylnaphthyl-1- 4-HO-benzyl Methyl Benzylamino 578 579 ylmethyl527 (S)-Methylnaphthyl-1- 4-HO-benzyl Methyl Benzylamino 578 579ylmethyl 528 Naphthyl-1-ylmethyl 3-Oxy-pyridin-1- Methyl Benzylamino 565566 ylmethyl 529 (R)-alpha 4-HO-benzyl Methyl Benzylamino 578 579methylbenzyl 530 Naphthyl-2-ylmethyl 4-HO-benzyl Methyl Benzylamino 564565 531 4-F-naphthyl-1- 4-HO-benzyl Methyl Benzylamino 582 583 ylmethyl532 2-Methoxybenzyl 4-HO-benzyl Methyl Benzylamino 544 545 5334-Cl-benzyl 4-HO-benzyl Methyl Benzylamino 548 549 534 3,4-Cl₂-benzyl4-HO-benzyl Methyl Benzylamino 582 583 535 2-CF₃Obenzyl 4-HO-benzylMethyl Benzylamino 598 599 536 2-CF₃Sbenzyl 4-HO-benzyl MethylBenzylamino 614 615 537 2-CF₃benzyl 4-HO-benzyl Methyl Benzylamino 582583 538 5-Quinoline-1yl- 4-HO-benzyl Methyl Benzylamino 565 566 methyl539 8-Quinoline-1yl- 3-t-Bu-4-HO-benzyl Methyl Benzylamino 621 622methyl 540 8-Quinoline-1yl- 4-NO₂-benzyl Methyl Benzylamino 594 595methyl 541 8-Quinoline-1yl- (1H-Pyrrol-2-yl)- Methyl Benzylamino 538 539methyl methyl 542 Naphthyl-1-ylmethyl 4-Benzyloxy- Methyl Benzylamino697 698 carbonylaminobenzyl 543 2,3-Cl₂-benzyl 4-HO-benzyl MethylBenzylamino 582 583 544 Pentafluorobenzyl 4-HO-benzyl Methyl Benzylamino604 605 545 Benzyl 4-HO-benzyl Methyl Benzylamino 514 515 546Quinoxaline-5yl- 4-HO-benzyl Methyl Benzylamino 566 567 methyl 5478-Quinoline-1yl- 3-Pyridylmethyl Methyl Benzylamino 550 551 methyl 5488-Quinoline-1 yl- Pentafluorobenzyl Methyl Benzylamino 639 640 methyl549 Naphthyl-1-ylmethyl 4-HO-benzyl Methyl Benzylamino(thiourea) 580 581550 Naphthyl-1-ylmethyl 4-Amino-benzyl Methyl Benzylamino 563 564 5513,4,5-tri- 4-Amino-benzyl Methyl Benzylamino 603 604 Methoxybenzyl 552Naphthyl-1-ylmethyl 4-Pyndylmethyl Methyl Benzylamino 549 550 553Naphthyl-1-ylmethyl (R) 4-HO-phenyl Methyl Benzylamino 550 551 5542-HO-3-Methoxy- 4-HO-benzyl Methyl Benzylamino 560 561 benzyl 555Naphthyl-1-ylmethyl 3-Nitro-4-HO- Methyl Benzylamino 609 610 benzyl 556Naphthyl-1-ylmethyl 4-CO₂H-CH₂O- Methyl Benzylamino 622 623 benzyl 557Naphthyl-1-ylmethyl 1-Naphthylamino- Methyl Benzylamino 641 642 methyl558 Naphthyl-1-ylmethyl 4-Oxy-pyridylmethyl Methyl Benzylamino 565 566559 4-F-alpha- 4-HO-benzyl Methyl Benzylamino 546 547 methylbenzyl 560Naphthyl-1-ylmethyl Benzoylaminoethyl Methyl Benzylamino 605 606 5618-Quinoline-1-yl- 3,4-(OH)2-benzyl Methyl Benzylamino 581 582 methyl 5624-N,N-Dimethyl- 4-HO-benzyl Methyl Benzylamino 557 558 aminobenzyl 563Naphthyl-1-ylmethyl (R) 4-F-benzyl Methyl Benzylamino 609 610 564Naphthyl-1-ylmethyl 4-HO-benzyl Methyl 2-Chloroethylamino 536 537 565Naphthyl-1-ylmethyl 4-HO-phenethyl Methyl Benzylamino 578 579 5664-F-benzyl 3-F,4-HO-benzyl Methyl Benzylamino 550 551 567 2,4-F₂-benzyl3-F,4-HO-benzyl Methyl Benzylamino 568 569 568 3-CF₃benzyl (R)4-HO-phenyl Methyl Benzylamino 568 569 569 (S)-Methylnaphthyl-1- (R)4-HO-phenyl Methyl Benzylamino 514 515 ylmethyl 570(R)-Methylnaphthyl-1- (R) 4-HO-phenyl Methyl Benzylamino 514 515ylmethyl 571 2,3,6-F₃-benzyl (R) 4-HO-phenyl Methyl Benzylamino 554 555572 3-F-benzyl (R) 4-HO-phenyl Methyl Benzylamino 518 519 5734-Cl-benzyl (R) 4-HO-phenyl Methyl Benzylamino 534 535 574 3-Cl-benzyl(R) 4-HO-phenyl Methyl Benzylamino 534 535 575 2-Cl-benzyl (R)4-HO-phenyl Methyl Benzylamino 534 535 576 3,4-Cl₂-benzyl (R)4-HO-phenylMethyl Benzylamino 568 569 577 3-CF₃O-benzyl (R) 4-HO-phenyl MethylBenzylamino 584 585 578 4-F-benzyl (R) 4-HO-phenyl Methyl Benzylamino518 519 579 2,4-F₂-benzyl (R) 4-HO-phenyl Methyl Benzylamino 536 537 5803-(2-Chloro-ethyl)- 4-HO-benzyl Methyl Benzylamino 634 635ureido]-benzyl 581 3-Aminobenzyl 4-HO-benzyl Methyl Benzylamino 529 530582 3-N- 4-HO-benzyl Methyl Benzylamino 543 544 Methylaminobenzyl 5833-N,N- 4-HO-benzyl Methyl Benzylamino 557 558 Dimethylaminobenzyl 5841H-Benzoimidazol-4- 4-HO-benzyl Methyl Benzylamino 554 555 ylmethyl 5852-HO-benzyl 4-HO-benzyl Methyl Benzylamino 530 531 586 2-Pyridylmethyl4-HO-benzyl Methyl Benzylamino 515 516 587 4-Pyridylmethyl 4-HO-benzylMethyl Benzylamino 515 516 588 8-quinolin-2-ylmethyl 4-HO-benzyl MethylBenzylamino 565 566 589 8-Benzofuran-4- 4-HO-benzyl Methyl Benzylamino554 555 ylmethyl 590 Naphthyl-1-ylmethyl 4-HO-phenyl Methyl Benzylamino550 551 591 4-F-benzyl 4-HO-phenyl Methyl Benzylamino 518 519 5922,4-F₂-benzyl 4-HO-phenyl Methyl Benzylamino 536 537 593(R)-Toluylmethyl 4-HO-benzyl Methyl Benzylamino 542 543 594(S)-Toluylmethyl 4-HO-benzyl Methyl Benzylamino 542 543 5951,2,3,4-tetrahydro- 4-HO-benzyl Methyl Benzylamino 554 555naphthalen-2-yl 596 Naphthyl-1-ylmethyl 3,4- Methyl Benzylamino 608 609Dimethoxybenzyl 597 2-Dimethylamino-6-F- 4-HO-benzyl Methyl Benzylamino575 576 benzyl 598 2- 4-HO-benzyl Methyl Benzylamino 557 558Dimethylaminobenzyl 599 Naphthyl-1-ylmethyl 4-CN-benzyl MethylBenzylamino 573 574 600 4-F-2-CF₃-benzyl 4-HO-benzyl Methyl Benzylamino599 600 601 4-Cl-2- 4-HO-benzyl Methyl Benzylamino 591 592Dimethylaminobenzyl 602 3-N,N- 4-HO-benzyl Methyl Benzylamino 571 572Ethylmethyllamino- benzyl 603 3-Diethylaminobenzyl 4-HO-benzyl MethylBenzylamino 585 586 604 4-Cl-3- 4-HO-benzyl Methyl Benzylamino 591 592Dimethylaminobenzyl 605 4-F-2- 4-HO-benzyl Methyl Benzylamino 575 576Dimethylaminobenzyl 606 3,5-(CH₃)₂)2- 4-HO-benzyl Methyl Benzylamino 585586 Dimethylamino-benzyl 607 3-(CH₃)-2- 4-HO-benzyl Methyl Benzylamino571 572 Dimethylaminobenzyl 608 6093,4-F₂-2- 4-HO-benzyl MethylBenzylamino 571 572 6-(CH₃)-2- Dimethylaminobenzyl 609 3,4-F₂-2-4-HO-benzyl Methyl Benzylamino 593 594 Dimethylaminobenzyl

TABLE 2B THE [4,4,0]REVERSE TURN MIMETICS LIBRARY

Mol. M + H No MOLSTRUCTURE Weight (MS) 802

480 481 803

430 431 804

416 417 805

464 465 806

430 431 807

430 431 808

448 448 809

416 417 810

431 432 811

446 447 812

450 451 813

515 516 814

582 583 815

532 533 816

518 519 817

566 567 818

532 533 819

532 533 820

550 551 821

518 519 822

534 535 823

548 549 824

552 553 825

617 618 826

542 543 827

492 493 828

478 479 829

526 527 830

492 493 831

492 493 832

510 511 833

478 479 834

494 495 835

508 509 836

512 513 837

577 578 838

468 469 839

516 517 840

482 483 841

482 483 842

468 469 843

484 485 844

498 499 845

502 503 846

567 568 847

508 509 848

458 459 849

444 445 850

492 493 851

458 459 852

458 459 853

476 477 854

444 445 855

460 461 856

474 475 857

478 479 858

543 544 859

494 495 860

444 445 861

430 431 862

478 479 863

444 445 864

444 445 865

462 463 866

430 431 867

446 447 868

460 461 869

464 465 870

529 530 871

558 559 872

508 509 873

494 495 874

542 543 875

508 509 876

508 509 877

526 527 878

494 495 879

510 511 880

524 525 881

528 529 882

593 594 883

432 433 884

480 481 885

446 447 886

446 447 887

464 465 888

432 433 889

447 448 890

462 463 891

466 467 892

531 532 893

558 559 894

508 509 895

494 495 896

542 543 897

508 509 898

508 509 899

526 527 900

494 495 901

510 511 902

524 525 903

528 529 904

593 594 905

544 545 906

494 495 907

480 481 908

528 529 909

494 495 910

494 495 911

512 513 912

480 481 913

496 497 914

510 511 915

514 515 916

579 580 917

464 465 918

450 451 919

498 499 920

464 465 921

464 465 922

482 483 923

450 451 924

466 467 925

480 481 926

484 485 927

549 550 928

480 481 929

430 431 930

416 417 931

464 465 932

430 431 933

430 431 934

448 449 935

416 417 936

431 432 937

446 447 938

450 451 939

515 516 940

504 505 941

454 455 942

440 441 943

488 489 944

454 455 945

454 455 946

472 473 947

440 441 948

455 456 949

470 471 950

474 475 951

539 540 952

604 605 953

554 555 954

540 541 955

588 589 956

554 555 957

554 555 958

572 573 959

540 541 960

556 557 961

570 571 962

574 575 963

639 640 964

528 529 965

478 479 966

464 465 967

512 513 968

478 479 969

478 479 970

496 497 971

464 465 972

480 481 973

494 495 974

498 499 975

563 564 976

582 583 977

532 533 978

518 519 979

566 567 980

532 533 981

532 533 982

551 552 983

518 519 984

534 535 985

548 549 986

552 553 987

618 619 988

482 483 989

432 433 990

418 419 991

466 467 992

432 433 993

432 433 994

450 451 995

418 419 996

433 434 997

447 448 998

452 453 999

517 518 1000

548 549 1001

498 499 1002

484 485 1003

532 535 1004

498 499 1005

498 499 1006

516 517 1007

484 485 1008

500 501 1009

514 515 1010

518 519 1011

583 584 1012

532 533 1013

518 519 1014

566 567 1015

532 533 1016

532 533 1017

551 552 1018

518 519 1019

534 535 1020

548 549 1021

552 553 1022

618 619 1023

528 529 1024

478 479 1025

464 465 1026

512 513 1027

478 479 1028

478 479 1029

496 497 1030

464 465 1031

480 481 1032

494 495 1033

498 499 1034

563 564 1035

528 529 1036

478 479 1037

464 465 1038

512 513 1039

478 479 1040

478 479 1041

496 497 1042

464 465 1043

480 481 1044

494 495 1045

498 499 1046

563 564 1047

556 557 1048

506 507 1049

492 493 1050

540 541 1051

506 507 1052

506 507 1053

524 525 1054

492 493 1055

508 509 1056

522 523 1057

526 527 1058

591 592 1059

546 547 1060

496 497 1061

482 483 1062

530 531 1063

496 497 1064

496 497 1065

514 515 1066

482 483 1067

498 499 1068

512 513 1069

516 517 1070

581 582 1071

528 529 1072

478 479 1073

464 465 1074

512 513 1075

478 479 1076

478 479 1077

496 497 1078

464 465 1079

480 481 1080

494 495 1081

498 499 1082

563 564 1083

514 515 1084

500 501 1085

548 549 1086

514 515 1087

514 515 1088

532 533 1089

500 501 1090

516 517 1091

530 531 1092

534 535 1093

599 600 1094

520 521 1095

470 471 1096

456 457 1097

504 505 1098

470 471 1099

470 471 1100

488 489 1101

456 457 1102

472 473 1103

486 487 1104

490 491 1105

555 556 1106

496 497 1107

482 483 1108

530 531 1109

496 497 1110

496 497 1111

514 515 1112

482 483 1113

498 499 1114

512 513 1115

516 517 1116

581 582 1117

542 543 1118

492 493 1119

478 479 1120

526 527 1121

492 493 1122

492 493 1123

510 511 1124

478 479 1125

494 495 1126

508 509 1127

512 513 1128

577 578 1129

550 551 1130

500 501 1131

486 487 1132

534 535 1133

500 501 1134

500 501 1135

518 519 1136

486 487 1137

501 502 1138

516 517 1139

520 521 1140

585 586 1141

588 589 1142

538 539 1143

524 525 1144

572 573 1145

538 539 1146

538 539 1147

556 557 1148

524 525 1149

540 541 1150

554 555 1151

558 559 1152

623 624 1153

508 509 1154

458 459 1155

444 445 1156

492 493 1157

458 459 1158

458 459 1159

476 477 1160

444 445 1161

460 461 1162

474 475 1163

478 479 1164

543 544 1165

618 619 1166

568 569 1167

554 555 1168

602 603 1169

568 569 1170

568 569 1171

586 587 1172

554 555 1173

570 571 1174

584 585 1175

588 589 1176

653 654 1177

494 495 1178

444 445 1179

430 431 1180

478 479 1181

444 445 1182

444 445 1183

462 463 1184

430 431 1185

446 447 1186

460 461 1187

464 465 1188

529 530 1189

506 507 1190

456 457 1191

442 443 1192

490 491 1193

456 457 1194

456 457 1195

474 475 1196

442 443 1197

458 459 1198

472 473 1199

476 477 1200

541 542 1201

592 593 1202

542 543 1203

528 529 1204

576 577 1205

542 543 1206

542 543 1207

561 562 1208

528 529 1209

544 545 1210

558 559 1211

562 563 1212

628 629 1213

538 539 1214

488 489 1215

474 475 1216

522 523 1217

488 489 1218

488 489 1219

506 507 1220

474 475 1221

490 491 1222

504 505 1223

508 509 1224

573 574 1225

510 511 1226

558 559 1227

524 525 1228

524 525 1229

510 511 1230

526 527 1231

540 541 1232

544 545 1233

609 610 1234

548 549 1235

498 499 1236

484 485 1237

532 533 1238

498 499 1239

498 499 1240

516 517 1241

484 485 1242

500 501 1243

514 515 1244

518 519 1245

583 584 1246

534 535 1247

484 485 1248

470 471 1249

518 519 1250

484 485 1251

484 485 1252

502 503 1253

470 471 1254

486 487 1255

500 501 1256

504 505 1257

569 570 1258

536 537 1259

486 487 1260

472 473 1261

520 521 1262

486 487 1263

486 487 1264

504 505 1265

472 473 1266

488 489 1267

502 503 1268

506 507 1269

571 572 1270

558 559 1271

508 509 1272

494 495 1273

542 543 1274

508 509 1275

508 509 1276

526 527 1277

494 495 1278

510 511 1279

524 525 1280

528 529 1281

593 594 1282

506 507 1283

456 457 1284

442 443 1285

490 491 1286

456 457 1287

456 457 1288

474 475 1289

442 443 1290

457 458 1291

472 473 1292

476 477 1293

541 542 1294

572 573 1295

522 523 1296

508 509 1297

556 557 1298

522 523 1299

522 523 1300

540 541 1301

508 509 1302

524 525 1303

538 539 1304

542 543 1305

607 608 1306

576 577 1307

526 527 1308

512 513 1309

560 561 1310

526 527 1311

526 527 1312

544 545 1313

512 513 1314

528 529 1315

542 543 1316

546 547 1317

611 612 1318

576 577 1319

526 527 1320

512 513 1321

560 561 1322

526 527 1323

526 527 1324

544 545 1325

512 513 1326

528 529 1327

542 543 1328

546 543 1329

611 612 1330

576 577 1331

526 527 1332

512 513 1333

560 561 1334

526 527 1335

526 527 1336

544 545 1337

512 513 1338

528 529 1339

542 543 1340

546 547 1341

611 612 1342

492 493 1343

442 443 1344

428 429 1345

476 477 1346

442 443 1347

442 443 1348

460 461 1349

428 429 1350

444 445 1351

458 459 1352

462 463 1353

527 528 1354

522 523 1355

472 473 1356

458 459 1357

506 507 1358

472 473 1359

472 473 1360

490 491 1361

458 459 1362

474 475 1363

488 489 1364

492 493 1365

557 558 1366

504 505 1367

454 455 1368

440 441 1369

488 489 1370

454 455 1371

454 455 1372

472 473 1373

440 441 1374

456 457 1375

470 471 1376

474 475 1377

539 540 1378

606 607 1379

556 557 1380

542 543 1381

590 591 1382

556 557 1383

556 557 1384

574 575 1385

542 543 1386

558 559 1387

572 573 1388

576 577 1389

641 642 1390

566 567 1391

516 517 1392

502 503 1393

550 551 1394

516 517 1395

516 517 1396

534 535 1397

572 573 1398

518 518 1399

532 533 1400

536 537 1401

601 602 1402

556 557 1403

506 507 1404

492 493 1405

540 541 1406

506 507 1407

506 507 1408

524 525 1409

492 493 1410

508 509 1411

522 523 1412

526 527 1413

591 592 1414

532 533 1415

582 583 1416

568 569 1417

516 517 1418

482 483 1419

582 583 1420

500 501 1421

468 469 1422

484 485 1423

498 499 1424

502 503 1425

567 568 1426

518 519 1427

568 569 1428

454 455 1429

502 503 1430

568 569 1431

468 469 1432

486 487 1433

454 455 1434

470 471 1435

484 485 1436

488 489 1437

553 554 1438

582 583 1439

532 533 1440

518 519 1441

566 567 1442

532 533 1443

532 533 1444

550 551 1445

518 519 1446

534 535 1447

548 549 1448

552 553 1449

617 618 1450

520 521 1451

470 471 1452

456 457 1453

504 505 1454

470 471 1455

470 471 1456

488 489 1457

556 557 1458

472 473 1459

486 487 1460

490 491 1461

555 556 1462

582 583 1463

532 533 1464

518 519 1465

566 567 1466

532 533 1467

532 533 1468

550 551 1469

518 519 1470

534 535 1471

548 549 1472

552 553 1473

517 618 1474

568 569 1475

518 519 1476

568 569 1477

552 553 1478

518 519 1479

518 519 1480

536 537 1481

504 505 1482

520 521 1483

534 535 1484

538 539 1485

603 604 1486

538 539 1487

488 489 1488

474 475 1489

522 523 1490

488 489 1491

488 489 1492

506 507 1493

474 475 1494

490 491 1495

504 505 1496

508 509 1497

573 574 1498

504 505 1499

454 455 1500

440 441 1501

488 489 1502

454 455 1503

454 455 1504

472 473 1505

540 441 1506

456 457 1507

470 471 1508

474 475 1509

439 540 1510

428 429 1511

478 479 1512

464 465 1513

512 513 1514

478 479 1515

478 479 1516

496 497 1517

464 465 1518

479 580 1519

494 495 1520

498 499 1521

463 564 1522

628 629 1523

578 579 1524

564 565 1525

612 613 1526

578 579 1527

578 579 1528

596 597 1529

564 565 1530

580 581 1531

594 595 1532

598 599 1533

663 664 1534

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474 475 2881

522 523 2882

488 489 2883

488 489 2884

506 507 2885

474 475 2886

490 491 2887

504 505 2888

508 509 2889

573 574 2890

648 649 2891

598 599 2892

584 585 2893

632 633 2894

598 599 2895

598 599 2896

616 617 2897

584 585 2898

600 601 2899

614 615 2900

618 619 2901

683 684 2902

622 623 2903

585 586 2904

619 620 2905

619 620 2906

585 586 2907

568 569 2908

583 584 2909

568 569 2910

462 463 2911

589 590 2912

589 590 2913

639 640 2914

571 572 2915

577 578 2916

617 618 2917

617 618 2918

583 584 2919

617 618 2920

617 618 2921

617 618 2922

599 600 2923

599 600 2924

639 640 2925

591 592 2926

591 592 2927

564 565 2928

554 555 2929

597 598 2930

659 660 2931

599 600 2932

599 600 2933

689 690 2934

569 570 2935

569 570 2936

571 572 2937

571 572 2938

633 634 2939

564 565 2940

571 572 2941

605 606 2942

608 609 2943

580 581 2944

605 606 2945

741 742 2946

550 551 2947

659 660 2948

625 626 2949

659 660 2950

554 555 2951

648 649 2952

659 660 2953

659 660 2954

659 660 2955

592 593 2956

667 668 2957

667 668 2958

565 566 2959

592 593 2960

592 593 2961

599 600 2962

667 668 2963

702 703 2964

688 689 2965

667 668 2966

512 513 2967

536 537 2968

659 660 2969

592 593 2970

592 593 2971

725 726 2972

617 618 2973

615 616 2974

588 589 2975

691 692 2976

566 567 2977

589 590 2978

571 572 2979

501 502 2980

599 600 2981

623 624 2982

552 553 2983

641 642 2984

579 580 2985

593 594 2986

613 614 2987

627 628 2988

605 606 2989

619 620 2990

625 626 2991

591 592 2992

617 618 2993

643 644 2994

667 668 2995

669 670 2996

555 556 2997

639 640 2998

637 638 2999

596 597 3000

581 582 3001

579 580 3002

625 626 3003

623 624 3004

659 660 3005

657 658 3006

595 596 3007

597 598 3008

669 670 3009

576 577 3010

574 575 3011

590 591 3012

611 612 3013

609 610 3014

611 612 3015

627 628 3016

639 640 3017

597 598 3018

623 624 3019

609 610 3020

681 682 3021

679 680 3022

578 579 3023

605 606 3024

611 612 3025

603 604 3026

605 606 3027

589 590 3028

808 809 3029

575 576 3030

605 606 3031

741 742 3032

618 619 3033

742 743 3034

539 540 3035

565 566 3036

565 566 3037

624 625 3038

541 542 3039

734 735 3040

575 576 3041

617 618 3042

566 567 3043

550 551 3044

647 648 3045

690 691 3046

555 556 3047

636 637 3048

664 665 3049

594 595 3050

655 656 3051

653 654 3052

578 579 3053

590 591 3054

577 578 3055

617 618 3056

576 577 3057

645 646 3058

609 610 3059

526 527 3060

528 529 3061

512 513 3062

542 543 3063

592 593 3064

597 598 3065

554 555 3066

554 555 3067

554 555 3068

639 640 3069

576 577 3070

598 599 3071

590 591 3072

590 591 3073

639 640 3074

639 640 3075

639 640 3076

583 584 3077

590 591 3078

579 580 3079

564 565 3080

569 570 3081

667 668 3082

564 565 3083

613 614 3084

721 722 3085

613 614 3086

579 580 3087

660 661 3088

568 569 3089

628 629 3090

584 585 3091

598 599 3092

667 668 3093

582 583 3094

624 625 3095

609 610 3096

570 571 3097

694 695 3098

694 695 3099

694 695 3100

694 695 3101

694 695 3102

694 695 3103

639 640 3104

615 616 3105

631 632 3106

680 681 3107

682 683 3108

637 638 3109

673 674 3110

689 690 3111

631 632 3112

615 616 3113

669 670 3114

640 641 3115

696 697 3116

611 612 3117

725 726 3118

612 613 3119

708 709 3120

615 616 3121

464 465 3122

478 479 3123

558 559 3124

572 573 3125

490 491 3126

504 505 3127

437 438 3128

541 542 3129

571 572 3130

599 600 3131

569 570 3132

567 568 3133

468 469 3134

453 454 3135

616 617 3136

574 575 3137

590 591 3138

688 689In addition, synthesis of the peptide mimetics of the library of thepresent invention may be accomplished using the General Scheme of[4,3,0] Reverse-Turn Mimetic Library as follows:

Synthesis of the peptide mimetics of the bicyclic template libraries ofthe present invention was accomplished using FlexChem Reactor Blockwhich has 96 well plate by known techniques. In the above scheme ‘Pol’represents Bromoacetal resin (Advanced ChemTech) and detailed procedureis illustrated below.

Step 1

The bromoacetal resin (1.6 mmol/g) and a solution of R1 amine in DMSO(2M solution) were placed in 96 well Robbins block (FlexChem). Thereaction mixture was shaken at 60° C. using rotating oven [RobbinsScientific] for 12 hours. The resin was washed with DMF, MeOH, and thenDCM

Step 2

A solution of commercial available Fmoc-Amino Acids (4 equiv.), PyBob (4equiv.), HOAt (4 equiv.), and DIEA (12 equiv.) in DMF was added to theresin. After the reaction mixture was shaken for 12 hours at roomtemperature, the resin was washed with DMF, MeOH, and then DCM.

Step 3

To the resin swollen by DMF before reaction was added 25% piperidine inDMF. After the reaction mixture was shaken for 30 min at roomtemperature. This deprotection step was repeated again and then washedwith DMF, Methanol, then DCM. A solution of hydrazine carbamoyl chloride(4 equiv.), HOBt (4 equiv.), and DIC (4 equiv.) in DMF was added to theresin. After the reaction mixture was shaken for 12 hours at roomtemperature, the resin was washed with DMF, MeOH, and then DCM.

Step 4

To the resin swollen by DMF before reaction was added 25% piperidine inDMF. After the reaction mixture was shaken for 30 min at roomtemperature. This deprotection step was repeated again and then washedwith DMF, Methanol, then DCM. To the resin swollen by DCM beforereaction was added R₁-isocynate (5 equiv.) in DCM. After the reactionmixture was shaken for 12 hours at room temperature the resin was washedwith DMF, MeOH, then DCM.

Step 5

The resin was treated with formic acid (1.2 mL each well) for 18 hoursat room temperature. After the resin was removed by filtration, thefiltrate was condensed under reduced pressure using SpeedVac [SAVANT] togive the product as oil. These products were diluted with 50%water/acetonitrile and then lyophilized after freezing.

Table 3 shows a [4,3,0] reverse turn mimetics library which can beprepared according to the present invention, of which representativepreparation is given in Example 5.

TABLE 3 THE [4,3,0] REVERSE TURN MIMETICS LIBRARY

Mol. No R₂ R₄ R₆ R₁ Weight M + H 610 Isoamyl 4-HO-phenyl Methyl Phenyl466 467 611 Isoamyl 4-HO-phenyl Methyl 4-Me-phenyl 480 481 612 Isoamyl4-HO-phenyl Methyl 3,5-Me₂-phenyl 494 495 613 Isoamyl 4-HO-phenyl Methyl4-MeO-phenyl 496 497 614 Isoamyl 4-HO-phenyl Methyl 4-CF₃-phenyl 534 535615 Isoamyl 4-HO-phenyl Methyl Cyclohexyl 472 473 616 Isoamyl4-HO-phenyl Methyl Benzyl 480 481 617 Isoamyl 4-HO-phenyl Methyl

494 495 618 Isoamyl 4-HO-phenyl Methyl 4-MeO-benzyl 510 511 619 Isoamyl4-HO-phenyl Methyl Phenethyl 494 495 620 Isoamyl 4-HO-phenyl MethylPentyl 460 461 621 Isoamyl 4-HO-phenyl Methyl Hexyl 474 475 622 Benzyl4-HO-phenyl Methyl Phenyl 486 487 623 Benzyl 4-HO-phenyl Methyl4-Me-phenyl 500 501 624 Benzyl 4-HO-phenyl Methyl 3,5-Me₂-phenyl 514 515625 Benzyl 4-HO-phenyl Methyl 4-MeO-phenyl 516 517 626 Benzyl4-HO-phenyl Methyl 4-CF₃-phenyl 554 555 627 Benzyl 4-HO-phenyl MethylCyclohexyl 492 493 628 Benzyl 4-HO-phenyl Methyl Benzyl 500 501 629Benzyl 4-HO-phenyl Methyl

514 515 630 Benzyl 4-HO-phenyl Methyl 4-MeO-benzyl 530 531 631 Benzyl4-HO-phenyl Methyl Phenethyl 514 515 632 Benzyl 4-HO-phenyl MethylPentyl 480 481 633 Benzyl 4-HO-phenyl Methyl Hexyl 494 495 634Naphth-1-ylmethyl 4-HO-phenyl Methyl Phenyl 536 537 635Naphth-1-ylmethyl 4-HO-phenyl Methyl 4-Me-phenyl 550 551 636Naphth-1-ylmethyl 4-HO-phenyl Methyl 3,5-Me₂-phenyl 564 565 637Naphth-1-ylmethyl 4-HO-phenyl Methyl 4-MeO-phenyl 566 567 638Naphth-1-ylmethyl 4-HO-phenyl Methyl 4-CF₃-phenyl 604 605 639Naphth-1-ylmethyl 4-HO-phenyl Methyl Cyclohexyl 542 543 640Naphth-1-ylmethyl 4-HO-phenyl Methyl Benzyl 550 551 641Naphth-1-ylmethyl 4-HO-phenyl Methyl

564 565 642 Naphth-1-ylmethyl 4-HO-phenyl Methyl 4-MeO-benzyl 580 581643 Naphth-1-ylmethyl 4-HO-phenyl Methyl Phenethyl 564 565 644Naphth-1-ylmethyl 4-HO-phenyl Methyl Pentyl 530 531 645Naphth-1-ylmethyl 4-HO-phenyl Methyl Hexyl 544 545 646 Cyclohexylmethyl4-HO-phenyl Methyl Phenyl 492 493 647 Cyclohexylmethyl 4-HO-phenylMethyl 4-Me-phenyl 506 507 648 Cyclohexylmethyl 4-HO-phenyl Methyl3,5-Me₂-phenyl 520 521 649 Cyclohexylmethyl 4-HO-phenyl Methyl4-MeO-phenyl 522 523 650 Cyclohexylmethyl 4-HO-phenyl Methyl4-CF₃-phenyl 560 561 651 Cyclohexylmethyl 4-HO-phenyl Methyl Cyclohexyl468 469 652 Cyclohexylmethyl 4-HO-phenyl Methyl Benzyl 506 507 653Cyclohexylmethyl 4-HO-phenyl Methyl

520 521 654 Cyclohexylmethyl 4-HO-phenyl Methyl 4-MeO-benzyl 536 537 655Cyclohexylmethyl 4-HO-phenyl Methyl Phenethyl 520 521 656Cyclohexylmethyl 4-HO-phenyl Methyl Pentyl 486 487 657 Cyclohexylmethyl4-HO-phenyl Methyl Hexyl 500 501 658 4-methylbenzyl 4-HO-phenyl MethylPhenyl 500 501 659 4-methylbenzyl 4-HO-phenyl Methyl 4-Me-phenyl 514 515660 4-methylbenzyl 4-HO-phenyl Methyl 3,5-Me₂-phenyl 528 529 6614-methylbenzyl 4-HO-phenyl Methyl 4-MeO-phenyl 530 531 6624-methylbenzyl 4-HO-phenyl Methyl 4-CF₃-phenyl 568 569 6634-methylbenzyl 4-HO-phenyl Methyl Cyclohexyl 506 507 664 4-methylbenzyl4-HO-phenyl Methyl Benzyl 514 515 665 4-methylbenzyl 4-HO-phenyl Methyl

528 529 666 4-methylbenzyl 4-HO-phenyl Methyl 4-MeO-benzyl 544 545 6674-methylbenzyl 4-HO-phenyl Methyl Phenethyl 528 529 668 4-methylbenzyl4-HO-phenyl Methyl Pentyl 494 495 669 4-methylbenzyl 4-HO-phenyl MethylHexyl 508 509 670 Methoxypropyl 4-HO-phenyl Methyl Phenyl 468 469 671Methoxypropyl 4-HO-phenyl Methyl 4-Me-phenyl 482 483 672 Methoxypropyl4-HO-phenyl Methyl 3,5-Me₂-phenyl 496 497 673 Methoxypropyl 4-HO-phenylMethyl 4-MeO-phenyl 498 499 674 Methoxypropyl 4-HO-phenyl Methyl4-CF₃-phenyl 536 537 675 Methoxypropyl 4-HO-phenyl Methyl Cyclohexyl 474475 676 Methoxypropyl 4-HO-phenyl Methyl Benzyl 482 483 677Methoxypropyl 4-HO-phenyl Methyl

496 497 678 Methoxypropyl 4-HO-phenyl Methyl 4-MeO-benzyl 512 513 679Methoxypropyl 4-HO-phenyl Methyl Phenethyl 496 497 680 Methoxypropyl4-HO-phenyl Methyl Pentyl 462 463 681 Methoxypropyl 4-HO-phenyl MethylHexyl 476 477 682 Phenethyl 4-HO-phenyl Methyl Phenyl 500 501 683Phenethyl 4-HO-phenyl Methyl 4-Me-phenyl 514 515 684 Phenethyl4-HO-phenyl Methyl 3,5-Me₂-phenyl 528 529 685 Phenethyl 4-HO-phenylMethyl 4-MeO-phenyl 530 531 686 Phenethyl 4-HO-phenyl Methyl4-CF₃-phenyl 568 569 687 Phenethyl 4-HO-phenyl Methyl Cyclohexyl 506 507688 Phenethyl 4-HO-phenyl Methyl Benzyl 514 515 689 Phenethyl4-HO-phenyl Methyl

528 529 690 Phenethyl 4-HO-phenyl Methyl 4-MeO-benzyl 544 545 691Phenethyl 4-HO-phenyl Methyl Phenethyl 528 529 692 Phenethyl 4-HO-phenylMethyl Pentyl 494 495 693 Phenethyl 4-HO-phenyl Methyl Hexyl 508 509 6942,2-bisphenylethyl 4-HO-phenyl Methyl Phenyl 576 577 6952,2-bisphenylethyl 4-HO-phenyl Methyl 4-Me-phenyl 590 591 6962,2-bisphenylethyl 4-HO-phenyl Methyl 3,5-Me₂-phenyl 604 605 6972,2-bisphenylethyl 4-HO-phenyl Methyl 4-MeO-phenyl 606 607 6982,2-bisphenylethyl 4-HO-phenyl Methyl 4-CF₃-phenyl 644 645 6992,2-bisphenylethyl 4-HO-phenyl Methyl Cyclohexyl 582 583 7002,2-bisphenylethyl 4-HO-phenyl Methyl Benzyl 586 587 7012,2-bisphenylethyl 4-HO-phenyl Methyl

604 605 702 2,2-bisphenylethyl 4-HO-phenyl Methyl 4-MeO-benzyl 620 621703 2,2-bisphenylethyl 4-HO-phenyl Methyl Phenethyl 604 605 7042,2-bisphenylethyl 4-HO-phenyl Methyl Pentyl 570 571 7052,2-bisphenylethyl 4-HO-phenyl Methyl Hexyl 584 585 706Naphth-1-ylmethyl Benzyl Methyl Phenyl 520 521 707 Naphth-1-ylmethylBenzyl Methyl 4-Me-phenyl 534 535 708 Naphth-1-ylmethyl Benzyl Methyl3,5-Me₂-phenyl 548 549 709 Naphth-1-ylmethyl Benzyl Methyl 4-MeO-phenyl550 551 710 Naphth-1-ylmethyl Benzyl Methyl 4-CF₃-phenyl 588 589 711Naphth-1-ylmethyl Benzyl Methyl Cyclohexyl 526 527 712 Naphth-1-ylmethylBenzyl Methyl Benzyl 534 535 713 Naphth-1-ylmethyl Benzyl Methyl

548 549 714 Naphth-1-ylmethyl Benzyl Methyl 4-MeO-benzyl 564 565 715Naphth-1-ylmethyl Benzyl Methyl Phenethyl 548 549 716 Naphth-1-ylmethylBenzyl Methyl Pentyl 514 515 717 Naphth-1-ylmethyl Benzyl Methyl Hexyl528 529 718 Naphth-1-ylmethyl

Methyl Phenyl 498 499 719 Naphth-1-ylmethyl

Methyl 4-Me-phenyl 512 513 720 Naphth-1-ylmethyl

Methyl 3,5-Me₂-phenyl 526 527 721 Naphth-1-ylmethyl

Methyl 4-MeO-phenyl 528 529 722 Naphth-1-ylmethyl

Methyl 4-CF₃-phenyl 566 567 723 Naphth-1-ylmethyl

Methyl Cyclohexyl 504 505 724 Naphth-1-ylmethyl

Methyl Benzyl 512 513 725 Naphth-1-ylmethyl

Methyl

526 527 726 Naphth-1-ylmethyl

Methyl 4-MeO-benzyl 542 543 727 Naphth-1-ylmethyl

Methyl Phenethyl 526 527 728 Naphth-1-ylmethyl

Methyl Pentyl 492 493 729 Naphth-1-ylmethyl

Methyl Hexyl 506 507 730 Naphth-1-ylmethyl Naphth-1-ylmethyl MethylPhenyl 570 571 731 Naphth-1-ylmethyl Naphth-1-ylmethyl Methyl4-Me-phenyl 584 585 732 Naphth-1-ylmethyl Naphth-1-ylmethyl Methyl3,5-Me₂-phenyl 598 599 733 Naphth-1-ylm6thyl Naphth-1-ylmethyl Methyl4-MeO-phenyl 600 601 734 Naphth-1-ylmethyl Naphth-1-ylmethyl Methyl4-CF₃-phenyl 638 639 735 Naphth-1-ylmethyl Naphth-1-ylmethyl MethylCyclohexyl 576 577 736 Naphth-1-ylmethyl Naphth-1-ylmethyl Methyl Benzyl584 585 737 Naphth-1-ylmethyl Naphth-1-ylmethyl Methyl

598 599 738 Naphth-1-ylmethyl Naphth-1-ylmethyl Methyl 4-MeO-benzyl 614615 739 Naphth-1-ylmethyl Naphth-1-ylmethyl Methyl Phenethyl 598 599 740Naphth-1-ylmethyl Naphth-1-ylmethyl Methyl Pentyl 564 565 741Naphth-1-ylmethyl Naphth-1-ylmethyl Methyl Hexyl 578 579 742Naphth-1-ylmethyl Cyclohexylmethyl Methyl Phenyl 526 527 743Naphth-1-ylmethyl Cyclohexylmethyl Methyl 4-Me-phenyl 540 541 744Naphth-1-ylmethyl Cyclohexylmethyl Methyl 3,5-Me₂-phenyl 554 555 745Naphth-1-ylmethyl Cyclohexylmethyl Methyl 4-MeO-phenyl 556 557 746Naphth-1-ylmethyl Cyclohexylmethyl Methyl 4-CF₃-phenyl 594 595 747Naphth-1-ylmethyl Cyclohexylmethyl Methyl Cyclohexyl 532 533 748Naphth-1-ylmethyl Cyclohexylmethyl Methyl Benzyl 540 541 749Naphth-1-ylmethyl Cyclohexylmethyl Methyl

554 555 750 Naphth-1-ylmethyl Cyclohexylmethyl Methyl 4-MeO-benzyl 570571 751 Naphth-1-ylmethyl Cyclohexylmethyl Methyl Phenethyl 554 555 752Naphth-1-ylmethyl Cyclohexylmethyl Methyl Pentyl 520 521 753Naphth-1-ylmethyl Cyclohexylmethyl Methyl Hexyl 534 535 754Naphth-1-ylmethyl 4-chlorobenzyl Methyl Phenyl 554 555 755Naphth-1-ylmethyl 4-chlorobenzyl Methyl 4-Me-phenyl 568 569 756Naphth-1-ylmethyl 4-chlorobenzyl Methyl 3,5-Me₂-phenyl 582 583 757Naphth-1-ylmethyl 4-chlorobenzyl Methyl 4-MeO-phenyl 584 585 758Naphth-1-ylmethyl 4-chlorobenzyl Methyl 4-CF₃-phenyl 622 623 759Naphth-1-ylmethyl 4-chlorobenzyl Methyl Cyclohexyl 560 561 760Naphth-1-ylmethyl 4-chlorobenzyl Methyl Benzyl 568 569 761Naphth-1-ylmethyl 4-chlorobenzyl Methyl

582 583 762 Naphth-1-ylmethyl 4-chlorobenzyl Methyl 4-MeO-benzyl 598 599763 Naphth-1-ylmethyl 4-chlorobenzyl Methyl Phenethyl 582 583 764Naphth-1-ylmethyl 4-chlorobenzyl Methyl Pentyl 548 549 765Naphth-1-ylmethyl 4-chlorobenzyl Methyl Hexyl 562 563 766Naphth-1-ylmethyl Methyl Methyl Phenyl 444 445 767 Naphth-1-ylmethylMethyl Methyl 4-Me-phenyl 458 459 768 Naphth-1-ylmethyl Methyl Methyl3,5-Me₂-phenyl 472 473 769 Naphth-1-ylmethyl Methyl Methyl 4-MeO-phenyl474 475 770 Naphth-1-ylmethyl Methyl Methyl 4-CF₃-phenyl 512 513 771Naphth-1-ylmethyl Methyl Methyl Cyclohexyl 450 451 772 Naphth-1-ylmethylMethyl Methyl Benzyl 458 459 773 Naphth-1-ylmethyl Methyl Methyl

472 473 774 Naphth-1-ylmethyl Methyl Methyl 4-MeO-benzyl 488 489 775Naphth-1-ylmethyl Methyl Methyl Phenethyl 472 473 776 Naphth-1-ylmethylMethyl Methyl Pentyl 438 439 777 Naphth-1-ylmethyl Methyl Methyl Hexyl452 453 778 Naphth-1-ylmethyl Isobutyl Methyl Phenyl 486 487 779Naphth-1-ylmethyl Isobutyl Methyl 4-Me-phenyl 500 501 780Naphth-1-ylmethyl Isobutyl Methyl 3,5-Me₂-phenyl 514 515 781Naphth-1-ylmethyl Isobutyl Methyl 4-MeO-phenyl 516 517 782Naphth-1-ylmethyl Isobutyl Methyl 4-CF₃-phenyl 554 555 783Naphth-1-ylmethyl Isobutyl Methyl Cyclohexyl 492 493 784Naphth-1-ylmethyl Isobutyl Methyl Benzyl 500 501 785 Naphth-1-ylmethylIsobutyl Methyl

514 515 786 Naphth-1-ylmethyl Isobutyl Methyl 4-MeO-benzyl 530 531 787Naphth-1-ylmethyl Isobutyl Methyl Phenethyl 514 515 788Naphth-1-ylmethyl Isobutyl Methyl Pentyl 480 481 789 Naphth-1-ylmethylIsobutyl Methyl Hexyl 494 495 790 Naphth-1-ylmethyl MethylthioethylMethyl Phenyl 504 505 791 Naphth-1-ylmethyl Methylthioethyl Methyl4-Me-phenyl 518 519 792 Naphth-1-ylmethyl Methylthioethyl Methyl3,5-Me₂-phenyl 532 533 793 Naphth-1-ylmethyl Methylthioethyl Methyl4-MeO-phenyl 534 535 794 Naphth-1-ylmethyl Methylthioethyl Methyl4-CF₃-phenyl 572 573 795 Naphth-1-ylmethyl Methylthioethyl MethylCyclohexyl 510 511 796 Naphth-1-ylmethyl Methylthioethyl Methyl Benzyl518 519 797 Naphth-1-ylmethyl Methylthioethyl Methyl

532 533 798 Naphth-1-ylmethyl Methylthioethyl Methyl 4-MeO-benzyl 548549 799 Naphth-1-ylmethyl Methylthioethyl Methyl Phenethyl 532 533 800Naphth-1-ylmethyl Methyithioethyl Methyl Pentyl 498 499 801Naphth-1-ylmethyl Methylthioethyl Methyl Hexyl 512 513

In a further aspect of this invention, the present invention providesmethods for screening the libraries for bioactivity and isolatingbioactive library members.

In yet another aspect, the present invention provides a method forcarrying out a binding assay. The method includes providing acomposition that includes a first co-activator, an interacting protein,and a test compound. The amino acid structure of the first co-activatorincludes a binding motif of LXXLL, LXXLI or FxxFF wherein X is any aminoacid. The method further includes detecting an alteration in bindingbetween the first co-activator and the interacting protein due to thepresence of the compound, and then characterizing the test compound interms of its effect on the binding.

The assay may be carried out by any means that can measure the effect ofa test compound on the binding between two proteins. Many such assaysare known in the art and can be utilized in the method of the presentinvention, including the so-called Two-Hybrid and Split-Hybrid systems.

The Two-Hybrid system, and various means to carry out an assay usingthis system, are described in, e.g., U.S. Pat. No. 6,410,245. TheSplit-Hybrid system has been described by, e.g., Hsiu-Ming Shiu et al.Proc. Natl. Acad. Sci. USA, 93:13896-13901, November 1996; and John D.Crispino, et al. Molecular Cell, 3:1-20, February 1999. In theSplit-Hybrid system, a fusion protein is utilized where protein X isfused to the lexA DNA binding domains (pLexA) and protein Y is fused tothe transcription activator VP16 (pSHM.1-LacZ). Interaction betweenlexA-X and VP16-Y leads to the expression of the Tetracycline repressorprotein (TetR). TetR prevents transcription of the HIS3 reporter gene,making the cells unable to grow on media lacking histidine. Disruptionof protein-protein interaction will restore the ability of the cells togrow on such media by shutting down expression of the tetracyclinerepressor. Accordingly, compounds of the present invention may be addedto the growing cells, and if the addition of the compound restores theability of the cells to grow on the media, the compound may be seen asan effective disruptor of the protein-protein interaction.

The yeast strains required to make the Split-Hybrid system work can beemployed with two hybrid LexA/VP16 constructs such as those described byStanley M. Hollenberg, et al. Molecular and Cellular Biology15(7):3813-3822, July 1995. A useful modification of the Split-Hybridsystem was utilized by Takemaru, K. I. and Moon, R. T. J. of Cell Biol;149:249-254, 2000.

Other assay formats are also suitable. For example, reporter gene assaysfor AP-1, ELISA, for example, blocking the production of IL-2 by aT-cell line after stimulation with CD3 and CD28 to look for inhibitorsof IL-2 transcription. Direct binding assays (between coactivators andtheir partners) can be performed by surface plasmon resonancespectroscopy (Biacore, Sweden, manufactures suitable instruments) orELISA.

Exemplary transcriptional regulators include, without limitation, VP16,VP64, p300, CBP, PCAF, SRC1 PvALF, AtHD2A and ERF-2. See, for example,Robyr et al. (2000) Mol. Endocrinol. 14:329-347; Collingwood et al.(1999) J. Mol. Endocrinol. 23:255-275; Leo et al. (2000) Gene 245:1-11;Manteuffel-Cymborowska (1999) Acta Biochim. Pol. 46:77-89; McKenna etal. (1999) J. Steroid Biochem. Mol. Biol. 69:3-12; Malik et al. (2000)Trends Biochem. Sci. 25:277-283; and Lemon et al. (1999) Curr. Opin.Genet Dev. 9:499-504. Other exemplary transcription factors include,without limitation, OsGAI, HALF-1, C1, AP1, ARF-5, -6, -7, and -8,CPRF1, CPRF4, MYC-RP/GP, and TRAB1. See, for example, Ogawa et al.(2000) Gene 245:21-29; Okanami et al. (1996) Genes Cells 1:87-99; Goffet al. (1991) Genes Dev. 5:298-309; Cho et al. (1999) Plant Mol. Biol.40:419-429; Ulmason et al. (1999) Proc. Natl. Acad. Sci. USA96:5844-5849; Sprenger-Haussels et al. (2000) Plant J. 22:1-8; Gong etal. (1999) Plant Mol. Biol. 41:33-44; and Hobo et al. (1999) Proc. Natl.Acad. Sci. USA 96:15, 348-15,353.

In a preferred embodiment, the transcriptional coactivator is a humantranscriptional coactivator. In another preferred embodiment, thetranscriptional coactivator is a member of the p300/CBP family ofco-activators which have histone acetyltransferase activity. p300 isdescribed for example by Eckner et al, 1994 and CBP by Bannister andKouzarides, 1996. For the purposes of the present invention, referenceto p300/CBP refers to human allelic and synthetic variants of p300, andto other mammalian variants and allelic and synthetic variants thereof,as well as fragments of said human and mammalian forms of p300. In oneaspect of the assay, the interacting protein is a transcription factoror a second co-activator.

In one aspect of the assay, the interacting protein is any one ofRIP140; SRC-1 (NCoA-1); TIF2 (GRIP-1; SRC-2); p (CIP; RAC3; ACTR; AIB-1;TRAM-1; SRC-3); CBP (p300); TRAPs (DRIPs); PGC-1; CARM-1; PRIP (ASC-2;AIB3; RAP250; NRC); GT-198; and SHARP (CoAA; p68; p72). In anotheraspect of the assay, the interacting protein is any one of TAL 1; p73;MDm2; TBP; HIF-1; Ets-1; RXR; p65; AP-1; Pit-1; HNF-4; Stat2; HPV E2;BRCA1; p45 (NF-E2); c-Jun; c-myb; Tax; Sap 1; YY1; SREBP; ATF-1; ATF-4;Cubitus; Interruptus; Gli3; MRF; AFT-2; JMY; dMad; PyLT: HPV E6; CITTA;Tat; SF-1; E2F; junB; RNA helicase A; C/EBP β; GATA-1; Neuro D;Microphthalimia; E1A; TFIIB; p53; P/CAF; Twist; Myo D; pp9O RSK; c-Fos;and SV40 Large T. In another aspect of the assay, the interactingprotein is any one of ERAP140; RIP140; RIP160; Trip1; SWI1 (SNF); ARA70;RAP46; TIF1; TIF2; GRIP1; and TRAP. In another aspect of the invention,the interacting protein is any one of VP16; VP64; p300; CBP; PCAF; SRC1PvALF; AtHD2A; ERF-2; OsGAI; HALF-1; C1; AP-1; ARF-5; ARF-6; ARF-7;ARF-8; CPRF1; CPRF4; MYC-RP/GP; and TRAB1. In another aspect of theinvention, the first co-activator is CBP or p300.

The test compound is selected from compounds as described herein. Forexample, compounds having the formula (I), (II), (III), (IV), (VI) and(VIa). Typically, a test compound will be evaluated at several differentconcentrations, where these concentrations will be selected, in part,based on the conditions of the assay, e.g., the concentrations of thefirst co-activator and the interacting protein. Concentrations in therange of about 0.1 to 10 μM are typical. In one aspect, the assayevaluates the relative efficacy of two compounds to affect the bindinginteraction between two proteins, where at least one of those twocompounds is a compound of the present invention. The more effectivecompound can than serve as a reference compound in a study of therelationship between compound structure and compound activity.

The libraries of the present invention were screened for bioactivity byvarious techniques and methods. In general, the screening assay may beperformed by (1) contacting the mimetics of a library with a biologicaltarget of interest, such as a receptor, to allow binding between themimetics of the library and the target to occur, and (2) detecting thebinding event by an appropriate assay, such as the calorimetric assaydisclosed by Lam et al. (Nature 354:82-84, 1991) or Griminski et al.(Biotechnology 12:1008-1011, 1994) (both of which are incorporatedherein by reference). In a preferred embodiment, the library members arein solution and the target is immobilized on a solid phase.Alternatively, the library may be immobilized on a solid phase and maybe probed by contacting it with the target in solution.

Table 4 below shows compounds for bioactivity test selected from thelibrary of the present invention and IC₅₀ values thereof, which aremeasured by the Reporter gene assay as described in Example 6.

TABLE 4 IC₅₀ (μM) OF SELECTED LIBRARY COMPOUNDS No STRUCTURE M.W. IC₅₀(μM) 1

580.7 12.8 2

579.6 12.6 3

632.5 13.9 4

617.6 11.8 5

564.6  6.8 6

564.6  6.1 7

564.6  2.2 8

531.6 14.5 9

531.6  6.7 10

531.6  4.0 11

531.6  4.6 12

549.6  9.0 13

549.6  6.4 14

549.6 17.7 15

581.6  4.2 16

567.6  3.8 17

548.0 14.3 18

548.0  3.3 19

582.5 11.5 20

527.6  5.1 21

527.6  5.0 22

543.6 10.4 23

573.6 10.7 24

563.7  5.0 25

581.6  3.0 26

543.6  7.1 27

543.6  5.2 28

548.0  7.5 29

582.5  3.8 30

597.6  7.5 31

613.7 11.9 32

581.6  4.1 33

564.6 13.0 34

565.6  4.4 35

579.7 11.4 36

549.6 12.5 37

545.6  2.3 38

556.7  7.1 39

564.6  9.7 40

553.6  7.0 41

541.6 13.6 42

574.7 18.2 43

556.7  5.2 44

599.6  1.3 45

591.1  2.2 46

570.7  4.4 47

584.7  3.5 48

570.7 10.9 49

592.6  1.4 50

574.6  1.3 51

584.7  4.8 52

621.69 25   53

584.72 9.0 ± 1.5 54

619.16 23.6 ± 5.6  55

584.72 7.2 ± 1.4 56

567.65 9.3 ± 1.6 57

582.70 9.4 ± 1.5 58

588.68 49.1 ± 8.1  59

588.68 5.3 ± 1.3 60

638.69 6.9 ± 1.7 61

570.69 25.6 62

616.73 9.7 ± 1.7 63

582.70 4.1 ± 0.5 64

616.73 25.3 ± 6.6  65

616.73  19 ± 7.1 66

598.74 11.8 67

598.74  6.8 68

590.68 4.3 ± 0.8 69

563.60 1.4 ± 0.7 70

553.62 8.8 ± 1.9 71

596.73 6.5 ± 0.7 72

658.76 1.6 ± 0.1 73

658.76  3.6 74

688.74 2.1 ± 0.2 75

568.64 50.5 ± 18.4 76

568.64 10.7 ± 2.5  77

570.67 7.2 ± 2.5 78

570.69 4.3 ± 0.9 79

632.76 16.5 ± 4.8  80

605.14 7.9 ± 2.0 81

607.61 66.1 ± 6.8  82

579.60 68.1 ± 8.9  83

605.14 46.4 ± 3.7  84

740.79 46.7 ± 6.7  85

549.67 15.6 ± 2.2  86

658.76 9.9 ± 2.6 87

624.74 8.1 ± 0.8 88

658.76 2.2 ± 0.2 89

553.62 13.9 ± 0.9  90

647.78  3.9 91

658.76 2.9 ± 0.2 92

658.76 3.8 ± 1.2 93

591.67 6.8 ± 1.3 94

666.78 7.6 ± 0.6 95

564.64 13.3 ± 1.4  96

591.67 8.1 ± 0.9 97

598.70 12.6 ± 1.2  98

666.78 14.4 ± 2.2  99

701.78 2.4 ± 0.3 100

666.78 2.7 ± 0.3 101

666.78  3.9 102

511.58 62.0 ± 17.0 103

535.59 14.5 ± 1.7  104

658.76 4.6 ± 0.4 105

591.67 16.6 ± 2.7  106

591.67 2.6 ± 0.2 107

724.82 2.7 ± 0.3 108

616.67 1.6 ± 0.1 109

616.67  2.1 110

615.13 3.8 ± 0.6 111

587.62 7.2 ± 0.8 112

690.80 4.1 ± 0.8 113

565.57 7.3 ± 1.1 114

588.67  0.4 ± 0.04 115

588.67  0.8 116

570.69 8.0 ± 0.7 117

598.70 6.9 ± 0.6 118

622.72 0.8 ± 0.1 119

551.60 8.8 ± 1.3 120

640.78 34.4 ± 4.9  121

578.67 3.0 ± 0.4 122

592.70 2.1 ± 0.4 123

612.73 11.7 ± 1.0  124

626.75 6.4 ± 0.4 125

605.14 9.8 ± 0.7 126

619.16 10.3 ± 1.5  127

624.74 1.8 ± 0.2 128

590.68 0.4 ± 0.1 129

617.15 2.4 ± 0.5 130

642.75 6.1 ± 0.4 131

666.78 2.2 ± 0.3 132

668.79 2.3 ± 0.5 133

638.77 3.5 ± 0.7 134

636.75 4.5 ± 0.9 135

595.65 2.4 ± 0.7 136

580.65 28.0 ± 2.9  137

625.13 0.6 ± 0.1 138

623.11 1.0 ± 0.2 139

659.18 1.1 ± 0.1 140

657.17 2.7 ± 0.3 141

594.69 1.8 ± 0.3 142

596.71 1.6 ± 0.4 143

575.61 1.3 ± 0.2 144

573.60 2.1 ± 0.2 145

610.71  0.3 ± 0.04 146

608.70 16.7 ± 1.4  147

610.71 9.4 ± 1.0 148

627.14 2.6 ± 0.3 149

639.15 31.0 ± 6.4  150

596.68 12.7 ± 0.7  151

596.68 9.2 ± 0.1 152

622.72 1.2 ± 0.3 153

622.72 1.9 ± 0.3 154

608.74 3.2 ± 0.4 155

680.77 30.5 ± 4.1  156

678.75 13.3 ± 1.6  157

577.63 4.2 ± 0.1 158

610.71  0.9 ± 0.02 159

602.64 2.7 ± 0.2 160

604.66 10.6 ± 0.5  161

741 1.8 ± 0.2 162

618 1.8 ± 0.6 163

742 1.7 ± 0.5 164

539 1.1 ± 0.2 165

565 3.9 ± 0.3 166

565 3.3 ± 0.2 167

624 1.3 ± 0.1 168

541 3.5 ± 0.3 169

734 1.0 ± 0.1 170

575 1.5 ± 0.5 171

617 44.7 ± 6.6  172

566 2.9 ± 0.4 173

690 1.8 ± 0.2 174

664 1.0 ± 0.1 175

594 5.4 ± 0.5 176

578 4.0 ± 0.4 177

590 5.3 ± 0.4 178

576 1.2 ± 0.1 179

645 2.3 ± 0.2 180

609 1.3 ± 0.1 181

592 6.7 ± 0.6 182

597  0.6 ± 0.04 183

554 12.8 ± 0.9  184

554 1.2 ± 0.1 185

639 23.4 ± 1.9  186

576 3.6 ± 0.3 187

598 1.1 ± 0.2 188

590 4.4 ± 0.2 189

639 8.7 ± 0.4 190

639 13.9 ± 0.8  191

639 5.2 ± 0.4 192

583 1.3 ± 0.3 193

569 2.5 ± 0.5 194

667 3.2 ± 0.4 195

564 22.3 ± 2.3  196

613 27.4 ± 2.8  197

721 0.7 ± 0.2 198

613 5.8 ± 0.3 199

660 1.0 ± 0.2 200

568 8.6 ± 0.4 201

628 5.8 ± 0.4 202

584 0.7 ± 0.1 203

598 0.7 ± 0.1 204

667 1.9 ± 0.1 205

582 3.5 ± 0.8 206

624 1.3 ± 0.1 207

609 1.5 ± 0.1 208

570 1.6 ± 0.4 209

694 1.9 ± 0.5 210

694 0.9 ± 0.1 211

694 2.3 ± 0.2 212

694 1.3 ± 0.3 213

694 1.7 ± 0.3 214

694 0.6 ± 0.2 215

639  18 ± 5.1 216

615 1.6 ± 0.2 (1.8 ± 0.3) 217

631 lower than  1.6 218

680 1.6 ± 1.2 219

682 2.2 ± 0.7 220

637 1.1 ± 0.3 221

673 4.7 ± 3.5 222

631 3.8 ± 2.7 223

615 0.6 ± 0.1 224

669 5.4 ± 1.0 225

611 0.1 ± 0.1

It has been found according to the present invention that compounds ofgeneral formula (I), and especially the compounds of general formula(VI), can inhibit CBP-mediated transcriptional activation in cancercells due to their specific binding to CBP. This conclusion is supportedby immunoprecipitation of CBP of SW480 cells with compounds of thepresent invention.

The compounds of the present invention can also inhibit the survivinexpression in SW480 cells, and therefore, inhibit the oncogenic activityin cancer cells. The compounds of the present invention can be used forinhibiting cancer cells, and thus, would be useful for the regulation ofcell growth. Supporting such results, the compounds of the presentinvention further shows that it can induce the caspase-3 activation inSW480 cells, and therefore, induce the apoptotic activity in cells. Thecompounds of the present invention can be also advantageously used forinducing apoptosis in cells.

To confirm the oncogenic activity in cancer cell in in vitro MTScytotoxicity assay was tested by following method.

(1) Cytotoxicity Test

SW480 or HCT116 cells were placed into 96 well microplate (10⁴cells/well) and incubated for 24 hours at 37° C. The cells were treatedwith TCF4 compound at various concentrations for 24 hours. 20 μl of MTSsolution (Promega) was added into each well and incubated for 2 hours at37° C. Cell viability was measured by reading the absorbance at 490 nmusing microplate reader (Molecular Device) and cytotoxicity of acompound at each concentration was calculated.

(2) Growth Inhibition Assay

SW480 or HCT116 cells were placed into 96 well microplate (10⁴cells/well) and incubated for 24 hours at 37° C. 20 μl of[3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium,inner salt](MTS) solution (Promega) was added into each well and theabsorbance after 2 hour incubation at 37° C. (negative control) wasread. And then, the cells were treated with TCF4 compound at variousconcentrations for 48 hours. 20 μl of MTS solution (Promega) was addedinto each well and incubated for 2 hour at 37° C. Cell viability wasmeasured by reading the absorbance at 490 nm using a microplate reader(Molecular device) and cytotoxicity of a compound at each concentrationwas calculated.

The results of oncogenic activity for selected library compounds wereshown in the Table 5. The compound numbers is Table 5 are unrelated tothe compound numbers in Table 4.

TABLE 5 ONCOGENIC ACTIVITY BY MTS OR SULFORHODAMINE B ASSAY FOR SELECTEDLIBRARY COMPOUNDS Growth Inhibition (GI50, uM) Compound Structure SW480HCT116 1

2.28 1.78 2

2.58 2.23 3

2.73 2.39 4

1.99 1.91 5

2.32 2.06 6

3.96 3.91 7

1.22 0.73 8

<0.3 <0.3 9

2.36 1.92 10

2.34 1.66 11

1.97 1.30 12

2.54 1.48 13

1.65 1.59 14

2.70 2.10 15

1.68 1.34 16

4.18 2.95 17

1.12 0.74 18

4.63 3.52 19

2.66 1.17 20

5.02 2.75 21

5.25 1.67 22

6.58 3.26 23

3.9 25.41 24

13.79 1.67 25

24.53 1.81 26

23.89 3.06 27

11.7 1.13 28

3.57 5.47 29

15.98 7.93 30

14.05 5.4 31

8.1 ± 0.7 5.0 ± 1.0 32

47.2 ± 12.1 16.9 ± 1.9  33

ND up to 50 uM 28.6 ± 2.0  34

13.8 ± 2.4  6.4 ± 1.3 35

4.7 ± 0.5 5.0 ± 0.7 36

21.9 ± 2.3  12.7 ± 1.3  37

10.4 ± 0.8  9.2 ± 0.9 38

8.5 6.9 39

22.8 ± 6.5  19.7 ± 3.3  40

6.4 ± 0.5 5.8 ± 0.4 41

34.4 ± 9.6  14.7 ± 2.6  42

24.7 10.8 43

ND up to 50 uM 39.1 44

3.8 ± 0.4 4.2 ± 0.5 45

2.5 ± 0.2 2.9 ± 0.4 46

5.5 ± 0.5 9.2 ± 0.9 47

6.2 12.2 48

20.7 ± 2.8  15.5 ± 2.3  49

1.4 ± 0.1 1.0 ± 0.2 50

4.6 2.6 51

3.0 ± 0.1 2.8 52

19.3 ± 2.1  9.7 ± 0.9 53

11.4 ± 0.9  4.7 ± 0.4 54

7.1 ± 0.5 4.9 ± 0.7 55

4.6 ± 0.5 4.1 ± 0.7 56

10.8 9.1 57

3.1 ± 0.3 5.1 ± 0.3 58

47.9 ± 7.2  22.3 ± 4.1  59

ND up to 50 uM 55.1 ± 33.7 60

8.3 ± 1.4 6.3 ± 2.6 61

11.3 ± 6.0  3.6 ± 0.3 62

35.3 ± 4.6  23.5 ± 2.7  63

18.8 ± 4.8  1.3 ± 0.1 64

12.0 ± 0.7  19.0 ± 1.6  65

7.3 4.7 66

3.0 ± 0.3 5.8 ± 0.3 67

0.6 ± 0.2  0.3 ± 0.03 68

3.7 ± 0.2 3.8 ± 0.6 69

17.9 ± 3.1  9.7 ± 1.0 70

7.4 ± 0.6 7.2 ± 0.7 71

4.6 ± 0.5 3.6 ± 0.7 72

10.9 ± 0.6  10.3 ± 1.6  73

9.2 ± 0.8 15.8 ± 2.6  74

1.3 ± 0.4 2.4 ± 0.3 75

2.0 ± 0.1 4.5 ± 0.4 76

4 6.1 77

26.5 ± 6.5  10.7 ± 0.8  78

2.2 ± 0.2 3.7 ± 0.3 79

2.8 ± 0.2 5.2 ± 0.4 80

4.0 ± 0.6 3.9 ± 0.6 81

0.5 ± 0.3 1.8 ± 0.1 82

1.5 1.4 83

2.3 ± 0.3 2.5 ± 0.1 84

8.4 ± 1.1 9.9 ± 1.0 85

1.4 ± 0.5 2.7 ± 0.3 86

9.6 ± 1.6 6.5 ± 0.6 87

0.6 ± 0.2 0.5 ± 0.1 88

0.3 0.4 89

14.6 ± 1.4  7.5 ± 1.0 90

12.6 ± 0.9  14.7 ± 1.0  91

1.5 ± 0.1 3.2 ± 0.2 92

12.9 ± 1.0  14.9 ± 2.2  93

1.9 ± 0.4 1.1 ± 0.1 94

1.1 ± 0.3  0.7 ± 0.07 95

16.2 ± 2.6  7.1 ± 1.2 96

3.7 ± 0.4 3.4 ± 0.4 97

7.1 ± 1.0 5.2 ± 0.5 98

7.0 ± 1.1 4.4 ± 0.5 99

 1.0 ± 0.05 0.7 ± 0.1 100

 0.3 ± 0.03 0.4 ± 0.1 101

 1.1 ± 0.07 0.9 ± 0.1 102

2.5 ± 0.4 4.9 ± 1.2 103

1.1 ± 0.1 1.5 ± 0.2 104

<0.4 <0.4 105

2.8 ± 0.2 2.1 ± 0.3 106

4.5 ± 0.3 2.8 ± 0.4 107

1.6 ± 0.1 1.6 ± 0.1 108

24.9 ± 2.2  37.9 ± 5.7  109

1.3 ± 0.3 1.1 ± 0.1 110

2.1 ± 0.3 1.9 ± 0.1 111

2.7 ± 0.8 2.1 ± 0.2 112

5.1 ± 0.5 4.7 ± 0.3 113

6.8 ± 1.4 3.7 ± 0.6 114

1.7 ± 0.7 1.9 ± 0.2 115

2.0 ± 0.7  1.1 ± 0.04 116

2.8 ± 0.9 1.7 ± 0.1 117

0.6 ± 0.1  0.3 ± 0.02 118

21.2 ± 1.5  23.2 ± 2.8  119

10.0 ± 1.3  9.5 ± 1.1 120

1.8 ± 0.2 2.6 ± 0.1 121

8.2 ± 0.5 13.1 ± 0.6  122

15.9 ± 5.2  14.8 ± 1.3  123

1.1 ± 0.3 1.7 ± 0.3 124

2.3 ± 0.2 1.4 ± 0.1 125

2.2 ± 0.3 1.9 ± 0.2 126

19.4 ± 3.0  11.6 ± 3.0  127

4.9 ± 0.7 4.3 ± 0.7 128

0.9 ± 0.1  1.0 ± 0.03 129

2.9 ± 0.5 3.1 ± 0.3 130

17.3 ± 1.2  10.7 ± 1.7  131

2.3 ± 0.1 1.7 ± 0.5 132

2.5 ± 0.1 2.2 ± 1.2 133

2.3 ± 0.1 2.1 ± 1.8 134

1.4 ± 0.4 0.8 ± 0.7 135

3.2 ± 0.4 3.1 ± 0.8 136

3.4 ± 0.4 3.0 ± 0.9 137

1.4 ± 0.4 1.3 ± 0.3 138

4.0 ± 0.4 3.9 ± 0.7 139

1.2 ± 0.1 1.0 ± 0.2 140

1.6 ± 0.5 1.7 ± 0.1 141

35 ± 11  21 ± 2.6 142

3.2 ± 0.3 3.3 ± 0.4 143

1.2 ± 0.1 1.2 ± 0.1 144

 0.5 ± 0.03 0.6 ± 0.1 145

6.4 ± 0.1 5.9 ± 0.3 146

3.7 ± 0.5 4.0 ± 0.5 147

6.1 ± 0.4 5.5 ± 0.4 148

1.3 ± 0.1 1.0 ± 0.3 149

2.3 ± 0.1 2.3 ± 0.4 150

1.3 ± 0.1 1.4 ± 0.2 151

11.2 ± 1.3  8.6 ± 0.9 152

0.7 ± 0.1 0.6 ± 0.1 153

12.8 ± 1.6  14.3 ± 7.0  154

0.7 ± 0.2 0.7 ± 0.2 155

26.3 ± 2.5  23.3 ± 1.2  156

3.7 ± 0.3 3.8 ± 0.2 157

1.0 ± 0.2 1.2 ± 0.1 58

4.4 ± 0.1 3.8 ± 0.5 159

9.1 ± 0.5 8.2 ± 0.4 160

13.7 ± 0.5  10.1 ± 01.3 161

4.2 ± 0.4 4.1 ± 0.5 162

1.0 ± 0.3 1.3 ± 0.7 163

2.4 ± 0.2 2.3 ± 0.4 164

3.0 ± 0.3 2.9 ± 0.4 165

22.8 ± 0.9  24.4 ± 1.9  166

27.9 ± 4.7  25.2 ± 3.2  167

 0.3 ± 0.02  0.2 ± 0.02 168

6.2 ± 0.8 6.5 ± 0.3 169

0.8 ± 0.1 1.0 ± 0.2 170

8.9 ± 0.8 8.6 ± 0.8 171

6.2 ± 1.0 6.0 ± 0.4 172

0.8 ± 0.1 0.9 ± 0.1 173

0.6 ± 0.1 0.8 ± 0.1 174

1.9 ± 0.2 1.8 ± 0.1 175

3.0 ± 0.4 2.5 ± 0.1 176

1.7 ± 0.2 1.7 ± 0.1 177

1.8 ± 0.1 1.6 ± 0.1 178

1.6 ± 0.2 1.5 ± 4.4 179

1.5 ± 0.1 1.6 ± 0.1 180

1.0 ± 0.1 1.1 ± 0.1 181

2.3 ± 0.1 2.3 ± 0.1 182

1.0 ± 0.1 0.8 ± 0.1 183

1.6 ± 0.3 1.5 ± 0.1 184

0.7 ± 0.4 0.7 ± 0.1 185

4.9 ± 0.4 4.5 ± 0.2 186

1.7 ± 0.1 2.0 ± 0.2 187

1.0 ± 0.1 1.0 ± 0.1 188

1.6 ± 0.2 1.8 ± 0.2 189

1.0 ± 0.1 1.2 ± 0.1 190

1.1 ± 0.3 1.0 ± 0.1 191

1.2 ± 0.1 1.4 ± 0.1 192

1.1 ± 0.1 1.3 ± 0.1 193

0.8 ± 0.1 1.1 ± 0.1 194

 0.2 ± 0.02  0.2 ± 0.02 195

3.1 ± 0.4 3.0 ± 0.3

In other aspects the present invention provides pharmaceuticalcompositions containing a compound having the general formula (I), orthe general formula (II), or the general formula (III), or the generalformula (IV), or the general formula (VI). These compositions may beused in various methods (e.g., treating cancer or Alzheimer's disease)of the present invention as described in detail below.

The pharmaceutical composition of the present invention is formulated tobe compatible with its intended route of administration. Examples ofroutes of administration include parenteral, e.g., intravenous,intradermal, subcutaneous, oral (e.g., inhalation), transdermal(topical), transmucosal, and rectal administration. Solutions orsuspensions used for parenteral, intradermal, or subcutaneousapplication can include the following components: a sterile diluent suchas water for injection, saline solution, fixed oils, polyethyleneglycols, glycerine, propylene glycol or other synthetic solvents;antibacterial agents such as benzyl alcohol or methyl parabens;antioxidants such as ascorbic acid or sodium bisulfite; chelating agentssuch as ethylenediaminetetraacetic acid; buffers such as acetates,citrates or phosphates and agents for the adjustment of tonicity such assodium chloride or dextrose. In addition, pH may be adjusted with acidsor bases, such as hydrochloric acid or sodium hydroxide. The parenteralpreparation can be enclosed in ampoules, disposable syringes or multipledose vials made of glass or plastic.

Pharmaceutical compositions suitable for injectable use include sterileaqueous solutions (where water soluble) or dispersions and sterilepowders for the extemporaneous preparation of sterile injectablesolutions or dispersion. For intravenous administration, suitablecarriers include physiological saline, bacteriostatic water, CremophorEL™ (BASF, Parsippany, N.J.) or phosphate buffered saline (PBS). In allcases, the composition must be sterile and should be fluid to the extentthat easy syringability exists. It must be stable under the conditionsof manufacture and storage and must be preserved against thecontaminating action of microorganisms such as bacteria and fungi. Thecarrier can be a solvent or dispersion medium containing, for example,water, ethanol, polyol (for example, glycerol, propylene glycol, andliquid polyethylene glycol, and the like), and suitable mixturesthereof. The proper fluidity can be maintained, for example, by the useof a coating such as lecithin, by the maintenance of the requiredparticle size in the case of dispersion and by the use of surfactants.Prevention of the action of microorganisms can be achieved by variousantibacterial and antifungal agents, for example, parabens,chlorobutanol, phenol, ascorbic acid, thimerosal, and the like. In manycases, it will be preferable to include isotonic agents, for example,sugars, polyalcohols such as manitol, sorbitol, sodium chloride in thecomposition. Prolonged absorption of the injectable compositions can bebrought about by including in the composition an agent which delaysabsorption, for example, aluminum monostearate and gelatin.

Sterile injectable solutions can be prepared by incorporating the activecompound (e.g., a compound having general formula (I), (II), (III),(IV), or (VI) in the required amount in an appropriate solvent with oneor a combination of ingredients enumerated above, as required, followedby filtered sterilization. Generally, dispersions are prepared byincorporating the active compound into a sterile vehicle that contains adispersion medium and the required other ingredients from thoseenumerated above. In the case of sterile powders for the preparation ofsterile injectable solutions, the preferred methods of preparation arevacuum drying and freeze-drying which yields a powder of the activeingredient plus any additional desired ingredient from a previouslysterile-filtered solution thereof.

Oral compositions generally include an inert diluent or an ediblecarrier. They can be enclosed in gelatin capsules or compressed intotablets. For the purpose of oral therapeutic administration, the activecompound can be incorporated with excipients and used in the form oftablets, troches, or capsules. Oral compositions can also be preparedusing a fluid carrier for use as a mouthwash, wherein the compound inthe fluid carrier is applied orally and swished and expectorated orswallowed. Pharmaceutically compatible binding agents, and/or adjuvantmaterials can be included as part of the composition. The tablets,pills, capsules, troches and the like can contain any of the followingingredients, or compounds of a similar nature: a binder such asmicrocrystalline cellulose, gum tragacanth or gelatin; an excipient suchas starch or lactose, a disintegrating agent such as alginic acid,Primogel, or corn starch; a lubricant such as magnesium stearate orSterotes; a glidant such as colloidal silicon dioxide; a sweeteningagent such as sucrose or saccharin; or a flavoring agent such aspeppermint, methyl salicylate, or orange flavoring.

For administration by inhalation, the compounds are delivered in theform of an aerosol spray from pressured container or dispenser thatcontains a suitable propellant, e.g., a gas such as carbon dioxide, or anebulizer.

Systemic administration can also be by transmucosal or transdermalmeans. For transmucosal or transdermal administration, penetrantsappropriate to the barrier to be permeated are used in the formulation.Such penetrants are generally known in the art, and include, forexample, for transmucosal administration, detergents, bile salts, andfusidic acid derivatives. Transmucosal administration can beaccomplished through the use of nasal sprays or suppositories. Fortransdermal administration, the active compounds are formulated intoointments, salves, gels, or creams as generally known in the art.

The compounds can also be prepared in the form of suppositories (e.g.,with conventional suppository bases such as cocoa butter and otherglycerides) or retention enemas for rectal delivery.

In one embodiment, the active compounds are prepared with carriers thatwill protect the compound against rapid elimination from the body, suchas a controlled release formulation, including implants andmicroencapsulated delivery systems. Biodegradable, biocompatiblepolymers can be used, such as ethylene vinyl acetate, polyanhydrides,polyglycolic acid, collagen, polyorthoesters, and polylactic acid.Methods for preparation of such formulations will be apparent to thoseskilled in the art. The materials can also be obtained commercially fromAlza Corporation and Nova Pharmaceuticals, Inc. Liposbmal suspensions(including liposomes targeted to infected cells with monoclonalantibodies to viral antigens) can also be used as pharmaceuticallyacceptable carriers. These can be prepared according to methods known tothose skilled in the art, for example, as described in U.S. Pat. No.4,522,811.

It is especially advantageous to formulate oral or parenteralcompositions in dosage unit form for ease of administration anduniformity of dosage. Dosage unit form as used herein refers tophysically discrete units suited as unitary dosages for the subject tobe treated; each unit containing a predetermined quantity of activecompound calculated to produce the desired therapeutic effect inassociation with the required pharmaceutical carrier. The specificationfor the dosage unit forms of the invention are dictated by and directlydependent on the unique characteristics of the active compound and theparticular therapeutic effect to be achieved, and the limitationsinherent in the art of compounding such an active compound for thetreatment of individuals.

Toxicity and therapeutic efficacy of such compounds can be determined bystandard pharmaceutical procedures in cell cultures or experimentalanimals, e.g., for determining the LD50 (the dose lethal to 50% of thepopulation) and the ED50 (the dose therapeutically effective in 50% ofthe population). The dose ratio between toxic and therapeutic effects isthe therapeutic index and it can be expressed as the ratio LD50/ED50.Compounds that exhibit large therapeutic indices are preferred. Whilecompounds that exhibit toxic side effects may be used, care should betaken to design a delivery system that targets such compounds to thesite of affected tissue in order to minimize potential damage touninfected cells and, thereby, reduce side effects.

The data obtained from the cell culture assays and animal studies can beused in formulating a range of dosage for use in humans. The dosage ofsuch compounds lies preferably within a range of circulatingconcentrations that include the ED50 with little or no toxicity. Thedosage may vary within this range depending upon the dosage formemployed and the route of administration utilized. For any compound usedin the method of the invention, the therapeutically effective dose canbe estimated initially from cell culture assays. A dose may beformulated in animal models to achieve a circulating plasmaconcentration range that includes the IC50 (i.e., the concentration ofthe test compound which achieves a half-maximal inhibition of symptoms)as determined in cell culture. Such information can be used to moreaccurately determine useful doses in humans. Levels in plasma may bemeasured, for example, by high performance liquid chromatography.

For instance, in certain embodiments, a pharmaceutical composition ofthe present invention is one suitable for oral administration in unitdosage form such as a tablet or capsule that contains from about 1 mg toabout 1 g of the compound of this invention. In some other embodiments,a pharmaceutical composition of the present invention is one suitablefor intravenous, subcutaneous or intramuscular injection. A patient mayreceive, for example, an intravenous, subcutaneous or intramuscular doseof about 1 μg/kg to about 1 g/kg of the compound of the presentinvention. The intravenous, subcutaneous and intramuscular dose may begiven by means of a bolus injection or by continuous infusion over aperiod of time. Alternatively a patient will receive a daily oral doseapproximately equivalent to the daily parenteral dose, the compositionbeing administered 1 to 4 times per day.

The following table illustrates representative pharmaceutical dosageforms containing the compound or pharmaceutically-acceptable saltthereof for therapeutics or prophylactic use in humans:

Tablet 1 mg/tablet Compound 100 Lactose Ph. Eur. 179 Croscarmellosesodium 12.0 Polyvinylpyrrolidone 6 Magnesium stearate 3.0 Tablet 2mg/tablet Compound 50 Lactose Ph. Eur. 229 Croscarmellose sodium 12.0Polyvinylpyrrolidone 6 Magnesium stearate 3.0 Tablet 3 mg/tabletCompound 1.0 Lactose Ph. Eur. 92 Croscarmellose sodium 4.0Polyvinylpyrrolidone 2.0 Magnesium stearate 1.0 Capsule mg/capsuleCompound 10 Lactose Ph. Eur. 389 Croscarmellose sodium 100 Magnesiumstearate 1.0 Injection I (50 mg/ml) Compound 0.5% w/v Isotonic aqueoussolution to 100%

The pharmaceutical composition containing the compound of generalformulae (I) or (II) or (III) or (IV) or (VI) can be used for treatmentof disorders modulated by Wnt signaling pathway, especially cancer, moreespecially colorectal cancer.

In one aspect, the present invention provides compounds that inhibit thebinding of a radiolabeled enkephalin derivative to the 6 and p opiatereceptors. Accordingly, the reverse-turn mimetics of the presentinvention may be used as receptor agonists and as potential analgesicagents.

In another aspect, the present invention provides methods for inhibitingtumor growth. Such methods comprise the step of administering to asubject (e.g., a mammalian subject) having a tumor a compound withgeneral formula (I), especially general formula (VI) in an amounteffective to inhibit tumor growth. A compound or composition inhibitstumor growth if the tumor sizes are statistically significantly smallerin subjects with the treatment of the compound or composition than thosewithout the treatment.

The inhibitory effect of a particular compound or composition of thepresent invention on tumor growth may be characterized by anyappropriate methods known in the art. For instance, the effect of thecompound or composition on survivin expression may be measured.Compounds or compositions down-regulate survivin expression are likelyto have inhibitory effects on tumor growth. In addition, assays usingtumor cell lines (e.g., soft agar assays using SW480 cells) and animalmodels for tumor growth (e.g., nude mice grafted with tumor cells andMin mouse model) may also be used to evaluate the inhibitory effect ontumor growth of a given compound or composition as described in detailin the examples. Other exemplary animal models or xenografts for tumorgrowth include those for breast cancer (Guo et al., Cancer Res. 62:4678-84, 2002; Lu et al., Breast Cancer Res. Treat. 57: 183-92, 1999),pancreatic cancer (Bouvet et al., Cancer Res. 62: 1534-40, 2002),ovarian tumor (Nilsson et al., Cancer Chemother. Pharmacol. 49: 93-100,2002; Bao et al., Gynecol. Oncol. 78: 373-9, 2000), melanoma (Demidem etal., Cancer Res. 61: 2294-300, 2001), colorectal cancer (Brown et al.,Dig. Dis. Sci. 45: 1578-84, 2000; Tsunoda et al., Anticancer Res. 19:1149-52, 1999; Cao et al., Clin. Cancer Res. 5: 267-74, 1999; Shawler etal., J. Immunother. Emphasis Tumor Immunol. 17: 201-8, 1995; McGregor etal., Dis. Colon. Rectum. 36: 834-9, 1993; Verstijnen et al., AnticancerRes. 8: 1193-200, 1988), hepatocellular cancer (Labonte et al., Hepatol.Res. 18: 72-85, 2000), and gastric cancer (Takahashi et al., Int. J.Cancer 85: 243-7, 2000).

The compound or composition that inhibits tumor growth may beadministrated into a subject with a tumor via an appropriate routedepending on, for example, the tissue in which the tumor resides. Theappropriate dosage may be determined using knowledge and techniquesknown in the art as described above. The effect of the treatment of thecompound or composition on tumor growth may also be monitored usingmethods known in the art. For instance, various methods may be used formonitoring the progression and/or growth of colorectal cancer, includingcolonoscopy, sigmoidoscopy, biopsy, computed tomograph, ultrasound,magnetic resonance imaging, and positron emission tomography. Methodsfor monitoring the progression and/or growth of ovarian cancer include,for example, ultrasound, computed tomography, magnetic resonanceimaging, chest X-ray, laparoscopy, and tissue sampling.

In a related aspect, the present invention provides a method fortreating or preventing cancer. Such methods comprise the step ofadministering to a subject in need thereof a compound or compositionhaving general formula (I), especially the compound of general formular(VI), in an amount effective to treat or prevent cancer in the subject.Treating cancer is understood to encompass reducing or eliminatingcancer progression (e.g., cancer growth and metastasis). Preventingcancer is understood to encompass preventing or delaying the onset ofcancer. Various types of cancer may be treated or prevented by thepresent invention. They include, but are not limited to, lung cancer,breast cancer, colorectal cancer, stomach cancer, pancreatic cancer,liver cancer, uterus cancer, ovarian cancer, gliomas, melanoma,lymphoma, and leukemia.

A subject in need of treatment may be a human or non-human primate orother animal with various types of cancer. A subject in need ofprevention may be a human or non-human primate or other animal that isat risk for developing cancer. Methods for diagnosing cancer andscreening for individuals with high risk of cancer are known in the artand may be used in the present invention. For instance, colorectalcancer may be diagnosized by fecal occult blood test, sigmoidoscopy,colonoscopy, barium enema with air contrast, and virtual colonoscopy. Anindividal with high risk of colorectal cancer may have one or morecolorectal cancer risk factors such as a strong family history ofcolorectal cancer or polyps, a known family history of hereditarycolorectal cancer syndromes, a personal history of adenomatous polyps,and a personal history of chronic inflammatory bowel disease.

A compound with general formula (I) useful in cancer treatment orprevention may be identified by appropriate methods known in the art.Methods that may be used to select compounds for inhibitory effect ontumor growth as described above may also be used. The route ofadministration, the dosage of a given compound, the effectiveness of thetreatment may be determined using knowledge and techniques known in theart. Factors that may be considered in making such a determinationinclude, for example, type and stage of the cancer to be treated.

The compound with general formula (I) useful in cancer treatment andprevention may be administered in combination with an anti-neoplasticagent. An anti-neoplastic agent refers to a compound that inhibits tumorgrowth. Exemplary anti-neoplastic agents include Fluorouracil;5-fluoro-2,4(1H, 3H)-pyrimidinedione (5-FU), taxol, cisplatin, mitomycinC, tegafur, raltitrexed, capecitabine, and irinotecan (Arango et al.,Cancer Research 61, 2001 4910-4915). A compound with general formula (I)administered in combination with an anti-neoplastic agent does notnecessarily require that the compound and the anti-neoplastic agent beadministered concurrently. The compound and the agent may beadministered separately as long as at a time point, they both haveeffects on same cancer cells.

In a further related aspect, the present invention provides methods forpromoting apoptosis in cancer cells. Such methods comprise the step ofcontacting cancer cells with a compound having general formula (I),especially a compound having general formula (VI), in an amounteffective to promote apoptosis in these cells. A compound promotesapoptosis if the number of cancer cells undergoing apoptosis isstatistically significantly larger in the presence of the compound thanthat in the absence of the compound. Such compounds may be identified bymethods known in the art (e.g., measuring caspase activities and/or celldeath) using cultured cancer cell lines, xenografts, or animal cancermodels. Preferably, the compound is more active in promoting apoptosisin cancer cells than in normal cells. Cancer cells treatable by thepresent method may be from various tissue origins.

In another aspect of the present invention, a method for treating adisorder modulated by Wnt signaling pathway in which the methodcomprises administering to a patient a safe and effective amount of thecompounds having general formula (I), especially the compound of generalformula (VI) is disclosed. Pharmaceutical composition containing thecompound of the present invention can be also used for this purpose. Inthis connection, it is found in the present invention that the compoundshaving general formula (I), especially the compound of general formula(VI) or the pharmaceutical composition containing thereof can be usefulfor the treatment of disorder modulated by TCF4-β catenin-CBP complex,which is believed to be responsible for initiating the overexpression ofcancer cells related to Wnt signaling pathway. Thus, it is anotheraspect of the present invention to provide a method for the treatment ofdisorder modulated by TCF4-β catenin-CBP complex, using the compoundshaving the general formula (I), especially the compound of generalformula (VI).

The present invention also provides compounds and methods for inhibitingsurvivin expression. Survivin is a target gene of the TCF/beta-cateninpathway, and more specifically is a target gene of theTCF/beta-catenin/CBP pathway. It is a member of the IAP (Inhibitor ofApoptosis Protein) family of proteins. Biological activity associatedwith survivin includes: highly expressed at G₂/M, regulating cell cycleentry and exit; associated with microtubule, centrosomes, centromeresand midbody depending upon the phases of the cell cycle; andanti-apoptosis via interacting directly or indirectly with caspases(e.g., caspase 3, 7 and 9). In connection with cancer, survivin iswidely and highly expressed in tumor cells, but expressed to little orno extent in normal tissue cells. Also, it has been observed that cancerpatients whose tumors expressed survivin had a decreased overallsurvival. Furthermore, the degree of surviving expression has beencorrelated with other cancer markers, e.g., Ki67, PNCA, p53, APC, etc.

The effect of a particular compound of the present invention on survivinexpression may be characterized by methods known in the art. Suchmethods include methods for characterizing survivin expression at thetranscriptional or translational level. Exemplary methods forcharacterizing survivin expression at the transcriptional level are:cDNA microarry, reverse transcription-polymerase chain reaction(RT-PCR), chromatin immunoprecipitation (ChIP), and assays for reporteractivities driven by survivin promoter. Exemplary methods forcharacterizing survivin expression at the translational level are:Western blot analysis, immunochemistry and caspase activities. Detaileddescriptions of the above exemplary methods may be found in the examplesbelow.

As described above, the present invention provides methods forinhibiting survivin expression. Such methods comprise the step ofcontacting a survivin-expressing cell with a compound of the presentinvention in an amount effective to inhibit survivin expression. Acompound inhibits survivin expression if survivin expression in a cellis decreased in the presence of the compound compared to survivinexpression in the absence of the compound. Survivin-expressing cellsinclude tumor cells that express, such as cells in or from lung cancer,breast cancer, stomach cancer, pancreatic cancer, liver cancer, uteruscancer, ovarian cancer, gliomas, melanoma, colorectal cancer, lymphomaand leukemia. The step of contacting the survivin-expressing cells withthe compound may be performed in vitro, ex vivo, or in vivo. A compounduseful in inhibiting survivin expression may be identified, and theeffects of a particular compound of the present invention may becharacterized, by appropriate methods known in the art, as described indetail above.

Compounds of the present invention have been shown to inhibit theexpression of survivin. Blanc-Brude et al., Nat. Medicine 8:987 (2002),have shown that survivin is a critical regulator of smooth muscle cellapoptosis which is important in pathological vessel-wall remodeling.Accordingly, another aspect of the present invention provides a methodof treating or preventing restenosis associated with angioplastycomprising administering to a subject in need thereof a safe andeffective amount of a reverse-turn mimetic of the present invention. Inone embodiment the invention treats the restenosis, i.e., administrationof a reverse-turn mimetic of the present invention to a subject havingrestenosis achieves a reduction in the severity, extent, or degree, etc.of the restenosis. In another embodiment the invention prevents therestenosis, i.e., administration of a reverse-turn mimetic of thepresent invention to a subject that is anticipated to develop new oradditional restenosis achieves a reduction in the anticipated severity,extent, or degree, etc. of the restenosis. Optionally, the subject is amammalian subject.

Compounds of the present invention have been shown to inhibitTCF/B-catenin transcription. Rodova et al., J. Biol. Chem. 277:29577(2002), have shown that PKD-1 promoter is a target of the B-catenin/TCFpathway. Accordingly, another aspect of the present invention provides amethod of treating or preventing polycystic kidney disease comprisingadministering to a subject in need thereof a safe and effective amountof a reverse-turn mimetic of the present invention. In one embodimentthe invention treats the polycystic kidney disease, i.e., administrationof a reverse-turn mimetic of the present invention to a subject havingpolycystic kidney disease achieves a reduction in the severity, extent,or degree, etc. of the polycystic kidney disease. In another embodimentthe invention prevents polycystic kidney disease, i.e., administrationof a reverse-turn mimetic of the present invention to a subject that isanticipated to develop new or additional polycystic kidney diseaseachieves a reduction in the anticipated severity, extent, or degree,etc. of the polycystic kidney disease. Optionally, the subject is amammalian subject.

Compounds of the present invention have been shown to inhibit theexpression of Wnt signaling. Hanai et al., J. Cell Bio. 158:529 (2002),have shown that endostatin, a known anti-angiogenic factor, inhibits Wntsignaling. Accordingly, another aspect of the present invention providesa method of treating or preventing aberrant angiogenesis diseasecomprising administering to a subject in need thereof a safe andeffective amount of a reverse-turn mimetic of the present invention. Inone embodiment the invention treats the aberrant angiogenesis disease,i.e., administration of a reverse-turn mimetic of the present inventionto a subject having aberrant angiogenesis disease achieves a reductionin the severity, extent, or degree, etc. of the aberrant angiogenesisdisease. In another embodiment the invention prevents aberrantangiogenesis disease, i.e., administration of a reverse-turn mimetic ofthe present invention to a subject that is anticipated to develop new oradditional aberrant angiogenesis disease achieves a reduction in theanticipated severity, extent, or degree, etc. of the aberrantangiogenesis disease. Optionally, the subject is a mammalian subject.

Compounds of the present invention have been shown to inhibit theexpression of Wnt signalling. Sen et al., P.N.A.S. (USA) 97:2791 (2000),have shown that mammals with rheumatoid arthritis demonstrate increasedexpression of Wnt and Fz in RA synovial tissue. Accordingly, anotheraspect of the present invention provides a method of treating orpreventing rheumatoid arthritis disease comprising administering to asubject in need thereof a safe and effective amount of a reverse-turnmimetic of the present invention. In one embodiment the invention treatsthe rheumatoid arthritis disease, i.e., administration of a reverse-turnmimetic of the present invention to a subject having rheumatoidarthritis disease achieves a reduction in the severity, extent, ordegree, etc. of the rheumatoid arthritis disease. In another embodimentthe invention prevents rheumatoid arthritis disease, i.e.,administration of a reverse-turn mimetic of the present invention to asubject that is anticipated to develop new or additional rheumatoidarthritis disease achieves a reduction in the anticipated severity,extent, or degree, etc. of the rheumatoid arthritis disease. Optionally,the subject is a mammalian subject.

Compounds of the present invention have been shown to inhibit theexpression of Wnt signalling. Uthoff et al., Int. J. Oncol. 19:803(2001), have shown that differential upregulation of disheveled and fz(Wnt pathway molecules) occurs in ulcerative colitis (compared toChron's disease patients). Accordingly, another aspect of the presentinvention provides a method of treating or preventing ulcerative colitiscomprising administering to a subject in need thereof a safe andeffective amount of a reverse-turn mimetic the present invention. In oneembodiment the invention treats the ulcerative colitis, i.e.,administration of a reverse-turn mimetic of the present invention to asubject having ulcerative colitis achieves a reduction in the severity,extent, or degree, etc. of the ulcerative colitis. In another embodimentthe invention prevents ulcerative colitis, i.e., administration of areverse-turn mimetic of the present invention to a subject that isanticipated to develop new or additional ulcerative colitis achieves areduction in the anticipated severity, extent, or degree, etc. of theulcerative colitis. Optionally, the subject is a mammalian subject.

Compounds of the present invention have been shown to inhibit WntTCF/catenin signalling. Accordingly, another aspect of the inventionprovides a method of treating or preventing tuberious sclerosis complex(TSC) comprising administering to a subject in need thereof a safe andeffective amount of a reverse-turn mimetic the present invention.Subjects having TSC typically develop multiple focal lesions in thebrain, heart, kidney and other tissues (see, e.g., Gomez, M. R. BrainDev. 17(suppl): 55-57 (1995)). Studies in mammalian cells have shownthat overexpression of TSC1 (which expresses hamartin) and TSC2 (whichexpresses tuberin) negatively regulates cell proliferation and inducesG₁/S arrest (see, e.g., Miloloza, A. et al., Hum. Mol. Genet. 9:1721-1727 (2000)). Other studies have shown that hamartin and tuberinfunction at the level of the p catenin degradation complex, and morespecifically that these proteins negatively regulate beta-cateninstability and activity by participating in the beta-catenin degradationcomplex (see, e.g., Mak, B. C., et al. J. Biol. Chem. 278(8): 5947-5951,(2003)). Beta-catenin is a 95-kDa protein that participates in celladhesion through its association with members of the membrane-boundcadherin family, and in cell proliferation and differentiation as a keycomponent of the Wnt/Wingless pathway (see, e.g., Daniels, D. L., etal., Trends Biochem. Sci. 26: 672-678 (2001)). Misregulation of thispathway has been shown to be oncogenic in humans and rodents. Thepresent invention provides compounds that modulate β-catenin activity,and particularly its interactions with other proteins, and accordinglymay be used in the treatment of TSC. Thus, in one embodiment theinvention treats TSC, i.e., administration of a reverse-turn mimetic ofthe present invention to a subject having TSC achieves a reduction inthe severity, extent, or degree, etc. of the TSC. In another embodimentthe invention prevents TSC, i.e., administration of a reverse-turnmimetic of the present invention to a subject that is anticipated todevelop new or additional TSC achieves a reduction in the anticipatedseverity, extent, or degree, etc. of the TSC. Optionally, the subject isa mammalian subject.

Compounds of the present invention have been shown to inhibit theexpression of Wnt signalling. The Kaposi's sarcoma-associatedherpesvirus (KSHV) latency-associated nuclear antigen (LANA) isexpressed in all KSHV-associated tumors, including Kaposi's sarcoma (KS)and β-cell malignancies such as primary effusion lymphoma (PEL) andmulticentric Castleman's disease. Fujimuro, M. et al., Nature Medicine9(3):300-306 (2003), have shown that LANA acts to stabilize β-catenin,apparently by redistribtution of the negative regular GSK-3β. Thepresent invention provides compounds and methods for inhibitingβ-catenin protein interactions, e.g., β-catenin/TCF complex formation.Thus, the compounds of the present invention thwart the LANA-inducedaccumulation of β-catenin/TCF complex and, at least in part, theconsequences of KSHV infection. Accordingly, another aspect of thepresent invention provides a method of treating or preventing conditionsdue to infection by Karposi's sarcoma-associated herpesvirus (KSHV).Such conditions include KSHV-associated tumors, including Kaposi'ssarcoma (KS) and primary effusion lymphoma (PEL). The method comprisesadministering to a subject in need thereof a safe and effective amountof a reverse-turn mimetic the present invention. In one embodiment theinvention treats the KSHV-associated tumor, i.e., administration of areverse-turn mimetic of the present invention to a subject having aKSHV-associated tumor achieves a reduction in the severity, extent, ordegree, etc. of the tumor. In another embodiment the invention preventsa KSHV-associated tumor, i.e., administration of a reverse-turn mimeticof the present invention to a subject that is anticipated to develop newor additional KSHV-associated tumors achieves a reduction in theanticipated severity, extent, or degree, etc. of the tumor. Optionally,the subject is a mammalian subject.

LEF/TCF DNA-binding proteins act in concert with activated β-catenin(the product of Wnt signaling) to transactivate downstream target genes.DasGupta, R. and Fuchs, E. Development 126(20):4557-68 (1999)demonstrated the importance of activated LEF/TCF complexes at distincttimes in hair development and cycling when changes in cell fate anddifferentiation commitments take place. Furthermore, in skinmorphogenesis, β-catenin has been shown to be essential for hairfollicle formation, its overexpression causing the “furry” phenotype inmice (Gat, U., et al. Cell 95:605-614 (1998) and Fuchs, E. Harvey Lect.94:47-48 (1999). See also Xia, X. et al. Proc. Natl. Aad. Sci. USA98:10863-10868 (2001). Compounds of the present invention have beenshown to inhibit the expression of Wnt signaling, and interfere withformation of β-catenin complexes. Accordingly, the present inventionprovides a method for modulating hair growth comprising administering toa subject in need thereof a safe and effective amount of a reverse-turnmimetic the present invention, where the amount is effective to modulatehair growth in the subject. Optionally, the subject is a mammaliansubject.

The present invention provides compounds useful in treating orpreventing Alzheimer's disease. Alzheimer's disease (AD) is aneurodegenerative disease with progressive dementia. This disease isaccompanied by three main structural changes in the brain, namely, i)intracellular protein deposits (also known as neurofibrillary tangles,or NFT), ii) extracellular protein deposits termed amyloid plaques thatare surrounded by dystrophic neuritis, and iii) diffuse loss of neurons.

The compounds or compositions of the present invention rescue defectesin neuronal differentiation caused by a presenilin-1 mutation and maydecrease the number, or rate at which neuronal precursor populationsdifferentiate to neurons in Alzheimer's brains. Presenilins aretransmembrane proteins whose functions are related to trafficking,turnover and cleavage of Notch and Amyloid Precursor Protein. Missensemutations in presenilin 1 (PS-1) are associated with early-onsetfamilial Alzheimer's disease (Fraser et al., Biochem. Soc. Symp. 67, 89(2001)). The compounds of the present invention may be applicable notonly to individuals with PS-1 familial Alzheimer's mutations, but alsoto general Alzheimer's patients.

In addition, the present invention provides a method for treating orpreventing Alzheimer's disease comprising administering to a subject inneed thereof a safe and effective amount of a reverse-turn mimetic ofthe present invention, where the amount is effective to treat or preventAlzheimer's disease in the subject. Treating Alzheimer's disease isunderstood to encompass reducing or eliminating the manifestation ofsymptoms characteistic of Alzheimer's disease, or delaying theprogression of this disease. Preventing Alzheimer's disease isunderstood to encompass preventing or delaying the onset of thisdisease.

A subject in need of treatment may be a human or non-human primate orother animal that is at various stages of Alzheimer's disease. Methodsfor diagnosing Alzheimer's disese are known in the art (see, e.g.,Dinsmore, J. Am. Osteopath. Assoc. 99(9 Suppl.):S1-6, 1999; Kurz et al.,J. Neural Transm. Suppl. 62: 127-33, 2002; Storey et al., Front Viosci.7: e155-84, 2002; Marin et al., Geriatrics 57: 36-40, 2002; Kril andHalliday, Int. Rev. Neurobiol. 48:167-217, 2001; Gurwitz, TrendsNeurosci. 23: 386, 2000; Muller-Spahn and Hock, Eur. Arch. PsychiatryClin. Neurosci. 249 Suppl. 3: 37-42; Fox and Rossor, Rev. Neuro. (Paris)155 Suppl. 4: S33-7, 1999), including the use of neuropsychologicalmeasures, functional imaging measures, biological markers, and autopsyof brain tissue. A subject in need of prevention may be a human ornon-human primate or other animal that is at risk for developingAlzheimer's disease, such as an individual having a mutation of certaingenes responsible for this disease (e.g., genes encoding amyloidprecursor protein, presenilin 1, and presenilin 2), and/or a geneinvolved in the pathogenesis of this disease (e.g., apolipoprotein Egene) (Rocchi et al., Brain Res. Bull. 61: 1-24, 2003).

Compounds with structures as set forth in formula (I) may be screenedfor their activities in treating or preventing Alzheimer's disease byany appropriate methods known in the art. Such screening may beinitially performed using in vitro cultured cells (e.g, PC-12 cells asdescribed in Example 8). Compounds capable of rescuing defects inneuronal differentiation caused by a presenilin 1 mutation may befurther screened using various animal models for Alzheimer's disease.Alternatively, compounds with structures as set forth in formula (I) maybe directedly tested in animal models for Alzheimer's disease. Manymodel systems are known in the art and may be used in the presentinvention (see, e.g., Rowan et al., Philos. Trans. R. Soc. Lond. B.Biol. Sci. 358: 821-8, 2003; Lemere et al., Neurochem. Res. 28: 1017-27,2003; Sant'Angelo et al., Neurochem. Res. 28: 1009-15, 2003; WeinerHarv. Rev. Psychiatry 4: 306-16, 1997). The effects of the selectedcompounds on treating or preventing Alzheimer's disease may becharacterized or monitored by methods known in the art for evaluatingthe progress of Alzheimer's disease, including those described above fordiagnosing this disease.

The present invention also provides methods for promoting neuriteoutgrowth. Such methods comprise the step of contacting a neuron with acompound according to formula (I) in an amount-effective to promoteneurite outgrowth. These methods are useful in treatingneurodegenerative diseases (e.g., glaucoma, macular degeneration,Parkinson's Disease, and Alzheimer's disease) and injuries to nervoussystem. A compound promotes neurite outgrowth if the neurite lengths ofneurons are statistically significantly longer in the presence of thecompound than those in the absence of the compound. Such a compound maybe identified using in vitro cultured cells (e.g, PC-12 cells,neuroblastoma B104 cell) (Bitar et al., Cell Tissue Res. 298: 233-42,1999; Pellitteri et al., Eur. J. Histochem. 45: 367-76, 2001; Satoh etal., Biochem. Biophys. Res. Commun. 258: 50-3-1999; Hirata and Fujisawa,J. Neurobiol. 32:415-25, 1997; Chauvet et al., Glia 18: 211-23, 1996;Vetter and Bishop, Curr. Biol. 5: 168-78, 1994; Koo et al., Proc. Natl.Acad. Sci. USA 90: 4748-52, 1993; Skubitz et al., J. Cell Biol.115:1137-48, 1991; O'Shea et al., Neuron 7: 231-7, 1991; Rydel andGreene, Proc. Natl. Acad. Sci. USA 85:1257-61, 1988) or using explants(Kato et al., Brain Res. 31: 143-7, 1983; Vanhems et al., Eur. J.Neurosci. 2: 776-82, 1990; Carri et al., Int. J. Dev. Neurosci. 12:567-78, 1994). Contacting a neuron with a compound according to thepresent invention may be carried out in vitro or in vivo. The resultingtreated neuron, if generated in vitro, may be transplanted into a tissuein need thereof (Lacza et al., Brain Res. Brain Res. Protoc. 11: 145-54,2003; Chu et al., Neurosci. Lett 343: 129-33, 2003; Fukunaga et al.,Cell Transplant 8: 435-41, 1999).

The present invention also provides methods for promotingdifferentiation of a neural stem cell comprising contacting a neuralstem cell with a compound according to formula (I) in an amounteffective to promote differentiation of a neural stem cell. Such methodsare also useful in treating neurodegenerative diseases (e.g., glaucoma,macular degeneration, Parkinson's Disease, and Alzheimer's disease) andinjuries to nervous system. “Neural stem cell” refers to a clonogenic,undifferentiated, multipotent cell capable of differentiating into aneuron, an astrocyte or an oligodendrocyte under appropriate conditions.A compound promotes differentiation of neural stem cells if neural stemcells exhibit a statistically significantly higher degree ofdifferentiation in the presence of the compound than in the absence ofthe compound. Such a compound may be identified using assays involvingin-vitro cultured stem cells or animal models (Albranches et al.,Biotechnol. Lett. 25: 725-30, 2003; Deng et al., Exp. Neurol. 182:373-82, 2003; Munoz-Elias et al., Stem Cells 21: 437-48, 2003; Kudo etal., Biochem. Pharmacol. 66: 289-95, 2003; Wan et al., Chin. Med. J.116: 428-31, 2003, Kawamorita et al., Hum. Cell 15: 178-82, 2002;Stavridis and Smith, Biochem. Soc. Trans. 31: 45-9, 2003; Pachernik etal., Reprod. Nutr. Dev. 42: 317-26, 2002; Fukunaga et al., supra). Theneural stem cell may be a cultured stem cell, a stem cell freshlyisolated from its source tissue, or a stem cell within its sourceorganism. Thus, contacting the neural stem cell with a compoundaccording to the present invention may be carried out either in vitro(for a cultured or freshly isolated stem cell) or in vivo (for a stemcell within its source organism). The resulting differentiated neuralcell, if generated in vitro, may be transplanted into a tissue in needthereof (Lacza et al., supra; Chu et al., supra; Fukunaga et al.,supra). Such a tissue includes a brain tissue or other nervous tissuethat suffers from a trauma or a neurodegenerative disease.

The following non-limiting examples illustrate the compounds,compositions, and methods of use of this invention.

EXAMPLES Preparation Example 1 Preparation Of(N-Fmoc-N′—R₃-hydrazino)-acetic acid

(1) Preparation of N-Fmoc-N′-Methyl Hydrazine

2 L, two-neck, round-bottomed-flask was fitted with a glass stopper anda calcium tube. A solution of methylhydrazine sulfate (20 g, 139 mmol,where R₃ is methyl) in THF (300 mL) was added and a solution of DiBoc(33 g, 153 mmol) in THF was added. Saturated sodium bicarbonate aqueoussolution (500 mL) was added dropwise via addition funnel over 2 hourswith vigorous stirring. After 6 hours, a solution of Fmoc-Cl (39 g, 153mmol) in THF was added slowly. The resulting suspension was stirred for6 hours at 0° C. The mixture was extracted with ethyl acetate (EA, 500mL) and the organic layer was retained. The solution was dried withsodium sulfate and evaporated in vacuo. The next step proceeded withoutpurification.

A 1 L, two-necked, round-bottom-flask was fitted with a glass stopperand a calcium tube. A solution of the product from the previous step inMeOH (300 mL) was added and conc. HCl (30 mL, 12 N) was added slowly viaaddition funnel with magnetic stirring in ice water bath and stirredovernight. The mixture was extracted with EA (1000 mL) and the organiclayer was retained. The solution was dried with sodium sulfate andevaporated in vacuo. The residue was purified by recrystallization withn-hexane and EA to give N-Fmoc-N′-methyl hydrazine (32.2 g, 83%). ¹HNMR(DMSO-D6) δ 7.90˜7.88 (d, J=6 Hz, 2H), δ 7.73˜7.70 (d, J=9 Hz, 2H),7.44˜7.31 (m, 4H), 4.52˜4.50 (d, J=6 Hz, 2H), 4.31˜4.26 (t, J=6 Hz, 1H),2.69 (s, 1H).

(2) Preparation of (N-Fmoc-N′-methyl-hydrazino)-acetic acid t-butylester

1 L, two-necked, round-bottom-flask was fitted with a glass stopper andreflux condenser connected to a calcium tube. A solution ofN-Fmoc-N′-methyl hydrazine (20 g, 75 mmol) in toluene (300 mL) wasadded. A solution of t-butylbromo acetate (22 g, 111 mmol) in toluene(50 mL) was added slowly. Cs₂CO₃ (49 g, 149 mmol) was added slowly. NaI(11 g, 74 mmol) was added slowly with vigorous stirring. The reactionmixture was stirred at reflux temperature over 1 day. The productmixture was filtered and extracted with EA (500 mL). The solution wasdried over sodium sulfate and evaporated in vacuo. The product waspurified by chromatography with hexane:EA=2:1 solution to give(N-Fmoc-N′-methyl-hydrazino)-acetic acid t-butyl ester (19.8 g, 70%).

¹H-NMR (CDCl₃-d) δ 7.78˜7.75 (d, J=9 Hz, 2H), δ 7.61˜7.59 (d, J=6 Hz,2H), 7.43˜7.26 (m, 4H), 4.42˜4.40 (d, J=6 Hz, 2H), 4.23 (b, 1H), 3.57(s, 2H), 2.78 (s, 3H), 1.50 (s, 9H).

(3) Preparation of (N-Fmoc-N′-methyl-hydrazino)-acetic acid

1 L, two-neck, round-bottomed-flask was fitted with a glass stopper andreflux condenser connected to a calcium tube.(N-Fmoc-N′-methyl-hydrazino)-acetic acid t-butyl ester (20 g, 52 mmol)was added. A solution of HCl (150 mL, 4 M solution in dioxane) was addedslowly with vigorous stirring in an ice water bath. The reaction mixturewas stirred at RT over 1 day. The solution was concentrated completelyunder reduced pressure at 40° C. A saturated aq. NaHCO₃ solution (100mL) was added and the aqueous layer was washed with diethyl ether (100mL). Conc. HCl was added dropwise slowly at 0° C. (pH 2-3). The mixturewas extracted and the organic layer was retained (500 mL, MC). Thesolution was dried with sodium sulfate and evaporated in vacuo. Theresidue was purified by recrystallization with n-hexane and ethylacetate to give (N-Fmoc-N′-methyl-hydrazino)-acetic acid (12 g, 72%).¹H-NMR (DMSO-d₆) δ 12.38 (s, 1H), 8.56 (b, 1H), 7.89˜7.86 (d, J=9 Hz,2H), 7.70˜7.67 (d, J=9 Hz, 2H), 7.43˜7.29 (m, 4H), 4.29˜4.27 (d, J=6 Hz,2H), 4.25˜4.20 (t, J=6 Hz, 1H), 3.47 (s, 2H), 2.56 (s, 3H).

Preparation Example 2 Preparation of (N-Moc-N′—R₇-hydrazino)-acetic acid

(1) Preparation of (N′-Methoxycarbonyl-hydrazino)-acetic acid ethylester

MOC—NH—NH₂ (50 g, 0.55 mol) was dissolved in DMF (300 ml), andthen-ethyl bromoacetate (68 ml, 0.555 mol) and potassium carbonate (77g, 0.555 mol) were added to the reaction vessel. The mixture was warmedto 50° C. for 5 hours. After the reaction was completed, the mixture wasfiltered, and diluted with EtOAc, and washed with brine (3 times). Thecrude product was purified by column (eluent: Hex/EtOAc=4/1) to provide72 of colorless oil.

(2) [N—R₇—N′-methoxycarbonyl-hydrazino]-acetic acid ethyl ester

The ethyl ester (10 g, 0.05 mol), potassium carbonate (6.9 g, 0.05 mol),and R₇-bromide (14.1 g, 0.06 mol) were dissolved in DMF (200 ml), andThe mixture was warmed to 50° C. for 5 hours. After the reaction wascompleted, the mixture was filtered, and diluted with EA, and washedwith brine (3 times). The crude product was purified by Chromatography(eluent: Hex/EtOAc=4/1).

(3) [N—R₇—N′-methoxycarbonyl-hydrazino]-acetic acid

The alkylated ethyl ester (9.5 g, 0.03 mol) was dissolved in THF/water(1/1, ml), and added 2N NaOH (28.3 ml) solution at 0° C. The mixture wasstirred at RT for 2 hours. After the starting ester was not detected onUV, the solution was diluted with EA, then separated. The aqueous layerwas acidified to pH 3˜4 by 1 N HCl, and the compound was extracted byDCM (3 times). The combined organic layer was dried over MgSO4, andevaporated to give a yellow solid.

Example 1

(1) Preparation of N^(β)-Moc-N^(α)-benzyl-hydrazinoglycine

This compound was prepared according to literature procedure.(Cheguillaume et. al., Synlett 2000, 3, 331)

(2) Preparation of1-Methoxycarbonyl-2,8-dibenzyl-6-methyl-4,7-dioxo-hexahydro-pyrazino[2,1-c][1,2,4]triazine

Bromoacetal resin (60 mg, 0.98 mmol/g) and a solution of benzyl amine inDMSO (2.5 ml, 2 M) were placed in vial with screw cap. The reactionmixture was shaken at 60° C. using rotating oven [Robbins Scientific]for 12 hours. The resin was collected by filtration, and washed withDMF, then DCM, to provide a first component piece.

A solution of Fmoc-alanine (4 equiv., commercially available, the secondcomponent piece), HATU (PerSeptive Biosystems, 4 equiv.), and DIEA (4equiv.) in NMP (Advanced ChemTech) was added to the resin. After thereaction mixture was shaken for 4 hours at room temperature, the resinwas collected by filtration and washed with DMF, DCM, and then DMF.

To the resin was added 20% piperidine in DMF. After the reaction mixturewas shaken for 8 min at room temperature, the resin was collected byfiltration and washed with DMF, DCM, and then DMF.

A solution of N^(β)-Moc-N^(α)-benzyl-hydrazinoglycine (4 equiv.,compound (3) in preparative example 2, where R₇ is benzyl, 3^(rd)component piece), HOBT [Advanced ChemTech] (4 equiv.), and DIC (4equiv.) in DMF was added to the resin prepared above. After the reactionmixture was shaken for 3 hours at room temperature, the resin wascollected by filtration and washed with DMF, DCM, and then MeOH. Theresin was dried in vacuo at room temperature.

The resin was treated with formic acid (2.5 ml) for 18 hours at roomtemperature. After the resin was removed by filtration, the filtrate wascondensed under reduced pressure to give the product as an oil. ¹H-NMR(400 MHz, CDCl₃) δ ppm; 1.51 (d, 3H), 2.99 (m, 1H), 3.39 (d, 1H), 3.69(m, 1H), 3.75 (m, 1H), 3.82 (s, 3H), 4.02 (d, 1H), 4.24 (d, 1H), 4.39(d, 1H), 4.75 (d, 1H), 5.14 (q, 1H), 5.58 (dd, 1H), 7.10-7.38 (m, 10H).

Example 2

(1) Preparation of N′-Fmoc-N-methyl-hydrazinocarbonyl chloride

An ice-cooled biphasic mixture of N-methyl hydrazine carboxylic acid9H-fluoren-9-ylmethyl ester (107 mg, 0.4 mmol) in 15 ml of CH₂Cl₂ and 15ml of saturated aq. NaHCO₃ was rapidly stirred while 1.93 M phosgene intoluene (1.03 ml, 2 mmol) was added as a single portion. The reactionmixture was stirred for 30 min, the organic phase was collected, and theaqueous phase was extracted with CH₂Cl₂. The combined organic layerswere dried over MgSO₄, filtered, and concentrated in vacuo to afford 128mg (97%) of carbamoyl chloride as a foamy solid. [Caution: Phosgenevapor is highly toxic. Use it in a hood]. This product was used for thefollowing solid phase synthesis without further purification.

(2) Preparation of2,5-Dimethyl-7-benzyl-3,6-dioxo-hexahydro-[1,2,4]triazolo[4,5-a]pyrazine-1-carboxylicacid benzylamide

Bromoacetal resin (30 mg, 0.98 mmol/g) and a solution of benzyl amine inDMSO (1.5 ml, 2 M) were placed in vial with screw cap. The reactionmixture was shaken at 60° C. using rotating oven [Robbins Scientific]for 12 hours. The resin was collected by filtration, and washed withDMF, then DCM, to provide the first component piece.

A solution of Fmoc-alanine (3 equiv., second component piece,commercially available), HATU (PerSeptive Biosystems, 3 equiv.), andDIEA (3 equiv.) in NMP (Advanced ChemTech) was added to the resin. Afterthe reaction mixture was shaken for 4 hours at room temperature, theresin was collected by filtration and washed with DMF, DCM, and thenDMF, to thereby add the second component piece to the first componentpiece.

To the resin was added 20% piperidine in DMF. After the reaction mixturewas shaken for 8 min at room temperature, the resin was collected byfiltration and washed with DMF, DCM, and then DMF.

A solution of N′-Fmoc-N-methyl-hydrazinocarbonyl chloride (combinedthird and fourth component pieces, 5 equiv.) obtained in the above step(1), DIEA (5 equiv.) in DCM was added to the resin prepared above. Afterthe reaction mixture was shaken for 4 hours at room temperature, theresin was collected by filtration and washed with DMF, DCM, and DMF.

To the resin was added 20% piperidine in DMF (10 ml for 1 g of theresin). After the reaction mixture was shaken for 8 min at roomtemperature, the resin was collected by filtration and washed with DMF,DCM, and then DMF.

The resin was treated with a mixture of benzyl isocyanate (4 equiv.) andDIEA (4 equiv.) in DCM for 4 hours at room temperature. Then, the resinwas collected by filteration and washed with DMF, DCM, and then MeOH.The resin was dried in vacuo at room temperature.

The resin was treated with formic acid for 14 hours at room temperature.After the resin was removed by filtration, the filtrate was condensedunder reduced pressure to give the product as an oil.

¹H-NMR (400 MHz, CDCl₃) δ ppm; 1.48 (d, 3H), 2.98 (s, 3H), 3.18 (m, 1H),3.46 (m, 1H), 4.37-4.74 (m, 5H), 5.66 (dd, 1H), 6.18 (m, 1H), 7.10-7.40(m, 10H).

Example 3 Preparation of2,5,7-Trimethyl-3,6-dioxo-hexahydro-[1,2,4]triazolo[4,5-a]pyrazine-1-carboxylicacid benzylamide

The title compound is prepared according to the same procedure asdescribed in Example 2, but reacting bromoacetal resin with a solutionof methyl amine instead of benzyl amine. ¹H-NMR (400 MHz, CDCl₃) δ ppm;1.48 (d, 3H), 2.99 (s, 3H), 3.03 (s, 3H), 3.38 (m, 1H), 3.53 (dd, 1H),4.36 (dd, 1H), 4.52 (q, 1H), 4.59 (dd, 1H), 5.72 (dd, 1H), 6.19 (br.t,1H), 7.10-7.38 (m, 5H).

Example 4 Preparation of2-Methyl-5-(p-hydroxyphenylmethyl)-7-naphthylmethyl-3,6-dioxo-hexahydro-[1,2,4]triazolo[4,5-a]pyrazine-1′-carboxylicacid benzylamide

Bromoacetal resin (30 mg, 0.98 mmol/g) and a solution of naphthylmethylamine in DMSO (1.5 ml, 2 M) were placed in vial with screw cap. Thereaction mixture was shaken at 60° C. using rotating oven [RobbinsScientific] for 12 hours. The resin was collected by filtration, andwashed with DMF, then DCM to provide the first component piece.

A solution of Fmoc-Tyr(OBut)-OH (3 equiv.), HATU (PerSeptive Biosystems,3 equiv.), and DIEA (3 equiv.) in NMP (Advanced ChemTech) was added tothe resin. After the reaction mixture was shaken for 4 hours at roomtemperature, the resin was collected by filtration and washed with DMF,DCM, and then DMF, to thereby add the second component piece to thefirst component piece.

To the resin was added 20% piperidine in DMF. After the reaction mixturewas shaken for 8 min at room temperature, the resin was collected byfiltration and washed with DMF, DCM, and then DMF.

A solution of N′-Fmoc-N-methyl-hydrazinocarbonyl chloride (5 equiv.),DIEA (5 equiv.) in DCM was added to the resin prepared above. After thereaction mixture was shaken for 4 hours at room temperature, the resinwas collected by filtration and washed with DMF, DCM, and DMF.

To the resin was added 20% piperidine in DMF (10 ml for 1 g of theresin). After the reaction mixture was shaken for 8 min at roomtemperature, the resin was collected by filtration and washed with DMF,DCM, and then DMF.

The resin was treated with a mixture of benzyl isocyanate (4 equiv.) andDIEA (4 equiv.) in DCM for 4 hours at room temperature. Then, the resinwas collected by filteration and washed with DMF, DCM, and then MeOH.The resin was dried in vacuo at room temperature.

The resin was treated with formic acid for 14 hours at room temperature.After the resin was removed by filtration, the filtrate was condensedunder reduced pressure to give the product as an oil.

¹H-NMR (400 MHz, CDCl₃) δ ppm; 2.80-2.98 (m, 5H), 3.21-3.37 (m, 2H),4.22-4.52 (m, 2H), 4.59 (t, 1H), 4.71 (d, 1H), 5.02 (dd, 1H), 5.35 (d,1H), 5.51 (d, 1H), 6.66 (t, 2H), 6.94 (dd, 2H), 7.21-8.21 (m, 12H).

Example 5 Preparation of2-Methyl-6-(p-hydroxyphenylmethyl)-8-naphthyl-4,7-dioxo-hexahydro-pyrazino[2,1-c][1,2,4]triazine-1-carboxylicacid benzylamide

Bromoacetal resin (60 mg, 0.98 mmol/g) and a solution of naphthyl aminein DMSO (2.5 ml, 2 M) were placed in vial with screw cap. The reactionmixture was shaken at 60° C. using rotating oven [Robbins Scientific]for 12 hours. The resin was collected by filtration, and washed withDMF, then DCM.

A solution of Fmoc-Tyr(OBut)-OH (4 equiv.), HATU [PerSeptive Biosystems](4 equiv.), and DIEA (4 equiv.) in NMP (Advanced ChemTech) was added tothe resin. After the reaction mixture was shaken for 4 hours at roomtemperature, the resin was collected by filtration and washed with DMF,DCM, and then DMF.

To the resin was added 20% piperidine in DMF. After the reaction mixturewas shaken for 8 min at room temperature, the resin was collected byfiltration and washed with DMF, DCM, and then DMF.

A solution of N^(β)-Fmoc-N^(α)-benzyl-hyrazinoglycine (4 equiv.), HOBT[Advanced ChemTech] (4 equiv.), and DIC (4 equiv.) in DMF was added tothe resin prepared above. After the reaction mixture was shaken for 3hours at room temperature, the resin was collected by filtration andwashed with DMF, and then DCM. To the resin was added 20% piperidine inDMF (10 ml for 1 g of the resin). After the reaction mixture was shakenfor 8 min at room temperature, the resin was collected by filtration andwashed with DMF, DCM, and then DMF.

The resin was treated with a mixture of benzyl isocyanate (4 equiv.) andDIEA (4 equiv.) in DCM for 4 hours at room temperature. Then, the resinwas collected by filteration and washed with DMF, DCM, and then MeOH.After the resin was dried in vacuo at room temperatur, the resin wastreated with formic acid (2.5 ml) for 18 hours at room temperature. Theresin was removed by filtration, and the filtrate was condensed underreduced pressure to give the product as an oil.

¹H-NMR (400 MHz, CDCl₃) δ ppm; 2.73 (s, 3H), 3.13 (d, 1H), 3.21-3.38 (m,3H), 3.55 (d, 1H), 3.75 (t, 1H), 4.22 (dd, 1H), 4.36 (dd, 1H), 4.79 (d,1H), 5.22 (t, 1H), 5.47 (m, 2H), 6.68 (d, 2H), 6.99 (d, 2H), 7.21-8.21(m, 12H); MS (m/z, ESI) 564.1 (MH⁺) 586.3 (MNa⁺).

Example 6 Bioassay for the Measurement of IC₅₀ Against SW480 Cells andCytotoxicity Test on the Cell Lines

The test compound (Compound A) used in this example was prepared inExample 4.

a. Reporter Gene Assay

SW480 cells were transfected with the usage of Superfect™ transfectreagent (Qiagen, 301307). Cells were trypsinized briefly 1 day beforetransfection and plated on 6 well plate (5×10⁵ cells/well) so that theywere 50-80% confluent on the day of transfection.

Four microgram (TOPFlash) and one microgram (pRL-null) of DNAs werediluted in 150 μl of serum-free medium, and 30 μl of Superfect™transfect reagent was added. The DNA-Superfect mixture was incubated atroom temperature for 15 min, and then, 1 ml of 10% FBS DMEM was added tothis complex for an additional 3 hours of incubation. While complexeswere forming, cells were washed with PBS twice without antibiotics.

The DNA-Superfec™ transfect reagent complexes were applied to the cellsbefore incubating at 37° C. at 5% CO₂ for 3 hours. After incubation,recovery medium with 10% FBS was added to bring the final volume to 1.18ml. After 3 hours incubation, the cells were harvested and reseeded to96 well plate (3×10⁴ cells/well). After overnight incubation at 37° C.at 5% CO₂, the cells were treated with Compound A for 24 hours. Finally,the activity was checked by means of luciferase assay (Promega, E1960).

FIG. 3 illustrates the results of the measurement of IC₅₀ of Compound Afor SW480 cells.

b. Sulforhodamine B (SRB) Assay

Growth inhibitory effect of Compound A on the cells listed below wasmeasured by the sulforhodamine B assay. SW480 cells in 100 μl media wereplated in each well of 96-well plate and allowed to attach for 24 hours.Compound A was added to the wells to produce the desired finalconcentrations, and the plates were incubated at 37° C. for 48 hours.The cells were then fixed by gentle addition of 100 μl of cold (4° C.)10% trichloroacetic acid to each well, followed by incubation at 4° C.for 1 hour. Plates were washed with deionized water five times andallowed to air dry. The cells were then stained by addition of 100 μlSRB solution (0.4% SRB (w/v) in 1% acetic acid (v/v)) to wells for 15min. After staining, the plates were quickly washed five times with 1%acetic acid to remove any unbound dye, and allowed to air dry. Bound dyewas solubilized with 10 mmol/L Tris base (pH 10.5) prior to reading theplates. The optical density (OD) was read on a plate reader at awavelength of 515 nm with Molecular Device. Inhibition of growth wasexpressed as relative viability (% of control) and GI₅₀ was calculatedfrom concentration-response curves after log/probit transformation.

Table 6 shows in vitro cyclotoxicity (SRB) assay data for Compound Aobtained in Example 4. The values in Table 6 are in μg/ml.

TABLE 6 Example Origin Cell 4 Cisplatin 5-FU Colon T84 1.134 >10 1.816LOVO 0.532 >10 1.029 HT29 1.694 >10 5.334 DLD-1 1.775 >10 >10 COLO2051.136 >10 1.130 CACO-2 1.201 >10 0.451 SW480-Kribb 1.137 >10 >10SW480-CWP 0.980 4.502 >10 SW620 1.426 >10 5.570 KM12 1.451 >10 2.729HCT15 2.042 >10 1.179 HCT116 0.96  >10 1.039 HCC2998 1.047 >10 5.486786-0 1.417 3.347 0.584 Leukemia HL60 1.243 >10 7.010 RPMI82261.1.177 >10 >10 K562/VIN 1.640 >10 7.071 K562/ADR 7.682 >10 >10 K5621.247 >10 6.133 Prostate PC3 1.207 >10 >10 HT1080 1.469 >10 0.798 LungA549 1.386 >10 1.007 NCI H460 1.498 >10 1.397 NCI H23 1.296 5.176 2.254Renal 293 0.731 6.641 2.015 CAKI-1 0.467 >10 0.925 ACHN 1.263 5.0195.062 Melanoma RPMI7951 0.936 5.010 0.920 M14 2.289 3.447 1.225 HMV-II4.834 3.190 0.695 HMV-I 1.153 5.478 2.110 G361 0.584 4.827 1.539 CRL1579 1.830 0.699 >10 A431 1.083 3.722 0.404 A253 1.398 2.084 2.926UACC62 0.563 >10 1.093 SK-MEL-28 1.291 >10 >10 SK-MEL-5 0.888 >10 2.434LOX-IMVI 1.526 >10 >10 A375 1.391 >10 1.464 Breast MCF7/ADR 9.4879.907 >10 MCF7 7.355 >10 1.751

Example 7 Min Mouse Model

Selected compounds of the present invention (Compound B and Compound C)were evaluated in the min mouse model to evaluate their efficacy asanit-cancer agents.

The min mouse model is a widely used model to test for this type ofefficacy. The numbers of polyp formed in small intestine and colon ofthese mice after various treatments were measured (Table 7). The datashown that both compounds, when administered at about 300 mpk, reducethe number of polyp in min mice compared to those in the control micetreated with vehicle only.

TABLE 7 MIN MOUSE MODEL DATA Polyp Number (Mean ± S.D.) % In- Small P(total) hibition Group Intestine Colon Total Vs. VH vs. VH Wild Type 0.0± 0.0 0.0 ± 0.0 0.0 ± 0.0 — — Vehicle 65.8 ± 15.9 1.8 ± 1.5 67.7 ± 15.3— — Compound C 69.2 ± 20.8 1.7 ± 1.5 71.4 ± 23.0 — — −100 mpk Compound C46.1 ± 17.1 1.1 ± 1.2 47.0 ± 16.9 <0.01 31 −300 mpk Compound B 45.2 ±22.1 1.4 ± 0.9 46.8 ± 17.0 <0.01 31 −300 mpk Sulindac 48.0 ± 20.7 0.5 ±0.5 48.5 ± 20.9 <0.05 28 −160 ppm

Example 8 Chemogenomic Inhibition of CBP/β-Catenin Interaction RescuesDefects in Neuronal Differentiation Caused by a Presenilin-1 Mutation

The following compound (Compound D) was used in this example:

Materials and Methods

Plasmids. TOPFLASH and FOPFLASH reporter constructs were transformedinto DH5α competent cells by standard protocol. Plasmids used fortransfection assays were isolated and purified using EndoFree Maxi Kit(Qiagen, Valencia, Calif.).

PC-12 Cell Culture. PC-12 cells were maintained in RPMI 1640supplemented with 10% horse serum, 5% fetal bovine serum, 4.5 g/Lglucose, 2 mM L-glutamine, 1.0 mM sodium pyruvate and 10 μg/mlpenicillin-streptomycin.

Cell Differentiation. Cell culture dishes were pre-coated overnight with0.25 mg/ml collagen (Cohesion, Calif), 10 μg/ml Poly-L-Lysine(Sigma-Aldrich, St. Louis, Mo.) and 12 μg/ml Polyethyleneimine (ICN, LaMesa, Calif.). Cells were cultured on coated dishes at 15,000 cells/cm²,and differentiated into a neuron-like phenotype by incubation in mediumwith reduced serum (1% fetal bovine serum), containing 50 ng/mlnerve-growth factor (NGF) (Sigma-Aldrich) for 10 days. NGF-containingmedium was changed every 2-3 days.

Treatment with Compound D. Compound D, a small molecule inhibitor ofβ-catenin/CBP interaction, was dissolved in DMSO at a stockconcentration of 100 mM. Differentiated PC-12/L286V cells were treatedwith increasing concentrations of this compound for 4 hours.Transfection was then initiated after this treatment period. For celldifferentiation experiments, Compound D was added at a concentration of10 μM, together with NGF, for the entire differentiation period.

Transfection. PC-12 cells were cultured and differentiated on 60-mmdishes. At the end of the 10-day differentiation period, cells weretransfected with 2 μg reporter constructs, TOPFLASH and FOPFLASH, per60-mm dish. Transfections were performed using Superfect (Qiagen)according to manufacturer's instructions.

Luciferase Assays. Cells were lysed, 6 hours after transfections, in 100μl of Cell Culture Lysis Reagent (Promega, Madison, Wis.), and scrapedinto microcentrifuge tubes. Tubes were then centrifuged briefly (about10 seconds) at 12000 rpm to pellet cell debris. Luciferase activity wasmeasured on 20 μl of cell lysate and 100 μl substrate from theLuciferase Assay System (Promega). Luciferase activity was measure usingPackard LumiCount. (Hewlett Packard). Quantitation of luciferase wasperformed in triplicates, and repeated in at least three independentexperiments.

Immunofluorescence. Cells were plated at a density of 10,000 cells/cm²on sterile coated 22×22 mm coverslips in a 6-well culture plate.Differentiation was initiated, as previously described, for 10 days. Thedifferentiated cells were then fixed in methanol for 15 minutes at −20°C. This is followed by a 15 minutes incubation with PBS+0.1% TritonX-100. The coverslips were incubated with antibodies raised againstEphrin B2 Receptor (Santa Cruz Biotechnology) and Gap-43 (NovusBiologicals) for 40 minutes at 37° C. After a series of washes withPBS-Triton X-100, secondary antibody conjugated to FITC (JacksonImmunoResearch, Westgrove, Pa.) was applied. All slides images wereacquired using a Nikon PCM2000 Laser Scanning Confocal Microscopemounted on a Nikon Eclipse E600 upright microscope (Nikon, Melville,N.Y.).

Quantitation of Neurite Outgrowth. Cell counts were taken from sixrandomly chosen microscopic fields (10×). In each field, total number ofcells, as well as cells that displayed neurites greater than twice thelength of the cell body was determined. The number of cells with suchoutgrowths was then expressed as a percentage of the total number ofcells. Values obtained were from duplicates of three independentexperiments.

RT-PCR. To analyze the mRNA levels for Ephrin B2 (EphB2) receptor, totalRNA was isolated using Trizol (Invitrogen-GIBCO-BRL, Baltimore, Md.)from differentiated cells. 2 μg RNA was reverse transcribed in a totalvolume of 20 μl with random hexamer (50 ng), and using the SuperscriptII reverse transcription system (Invitrogen-GIBCO-BRL), according tomanufacturer's guidelines. PCR was carried out in a 50 μl volumecontaining 5 μl cDNA, 100 pmol primers, 100 μM dNTPs, 1× Taq buffer and1.5 mM MgCl₂. Reaction mixtures were heated to 80° C. for 10 min, afterwhich Taq was added. cDNAs were amplified for 25 (EphB2 receptor) or 15(GAPDH) cycles. One round of amplification consisted of 1 min at 94° C.,2 min at 60° C., and 2 min at 72° C., with a final extension time of 10min at 72° C. The PCR products were resolved and visualized byelectrophoresis in a 2% gel, stained with ethidium bromide. EphB2receptor PCR primers used were, 5′-CACTACTGGACCGCACGATAC-3′ and5′-TCTACCGACTGGATCTGGTTCA-3′. Primer pairs for GAPDH were5′-GGTGCTGAGTATGTCGTGGA-3′ and 5′-ACAGTGTTCTGGGTGGCAGT-3′.

Results

Rat PC-12 cells are derived from the neural crest lineage and upon nervegrowth factor (NGF) treatment, undergo differentiation to aneurite-bearing sympathetic-like neuron (Greene and Tischler, Proc NatlAcad Sci USA 73, 2424 (1976)). Utilizing a PC-12 cell based model, theeffects of an early-onset FAD associated PS-1 mutation, PS-1/L286V, onTCF/β-catenin mediated transcription and neuronal differentiation werecharacterized. It has been demonstrated that specifically blockingtranscription mediated by TCF/β-catenin/CBP alleviates PS-1 induceddefects in neuronal differentiation.

PC-12 cells stably overexpressing either wild type PS-1 (PS-1/WT) ormutant PS-1 (PS-1/L286V) and a vector-transfected control cell line (Guoet al., Neuroreport, 8, 379 (1996)) were plated on dishes coated withcollagen, poly-L-lysine and poly-etheleneimine. Differentiation wasinduced by treatment with 50 ng/ml of NGF for 10 days. OverexpressingPS-1/WT cells or the vector-transfected cells had extensive neuriteformation (similar to PC-12 cell clones from ATCC), whereas thePS-1/L286V mutant cells had only stubby neurite formation (FIG. 4 A-C).Additionally, vector-transfected PC-12 control and PS-1/WT cellsdisplayed extensive expression of the neuronal differentiation markerGAP-43 (Gorgels et al., Neurosci Lett. 83, 59(1987)) (FIG. 4 D,E),whereas the PS-1/L286V cells were essentially devoid of this marker(FIG. 4 F).

To assess the effects of the PS-1/L286V mutation on canonicalWnt/β-catenin signaling, we transiently transfected NGF treated PC-12cells with Topflash, a Wnt/β-catenin signaling reporter construct (Morinet al., Science 275, 1787(1997)). As seen in FIG. 4F, the overexpressingPS-1/WT cells had similar levels of TCF/β-catenin signaling compared tothe vector control cells. However, the PS-1/L286V mutant cells displayedsignificantly (10-fold) increased Topflash expression. In contrast, thenegative control reporter construct Fopflash did not show anysignificant differences.

It was hypothesized that dysregulated TCF/β-catenin signaling in thePS-1/L286V mutant cells was responsible for the defectivedifferentiation and neurite outgrowth. To test this hypothesis, aspecific small molecule inhibitor of TCF/β-catenin signaling, Compound D(Emami et al., Cancer Cell, in press), was used. This small moleculeselectively blocks the β-catenin/CBP interaction, but not theβ-catenin/p300 interaction, thereby interrupting a subset ofTCF/β-catenin transcription. Treatment of the PS-1/L286V mutant cellswith 10 μM Compound D plus NGF decreased TCF/β-catenin reporter genetranscription, and led to essentially normal neurite outgrowth anddifferentiation (FIG. 5 A), similar to that seen in the overexpressingPS-1/WT cells (FIGS. 5 A, B), as compared to the untreated cells (FIG. 4C). Furthermore, PS-1/L286V mutants treated with Compound D showedsimilar intense GAP-43 staining to the PS-1/WT and vector-transfectedcells (FIG. 4 B). To demonstrate that Compound D treated mutant cellsdevelop neurites similar to that of the vector control or PS-1/WT cells,cells that had neurites greater than twice the length of the cell bodywere counted. Treatment with Compound D substantially increased thepercentage of cells bearing neurites to levels similar to that of thevector-transfected and overexpressing PS-1/WT cells (FIG. 5 C). It isconcluded that blocking transcription mediated by TCF/β-catenin/CBPcorrects many of the phenotypic defects in neurite outgrowth andneuronal differentiation due to the PS-1/L286V mutation.

Ephrin B2 receptors (EphB2) have been implicated in synapse formation(Wilkinson, Nat. Rev. Neurosci. 2, 155 (2001)) and the Ephrin A familyhas recently been shown to play a role in hippocampal dendritic spinemorphology (Murai et al., Nat Neurosci. 6, 153 (2003)). Focused EphB2expression was observed, which localized with neuronal processes in thevector and PS-1/WT-transfected cells (FIG. 6 A, B), whereas thePS-1/L286V mutant cells demonstrated very weak and diffuse EphB2 signal(FIG. 6 C). Increased TCF/β-catenin signaling in PS-1/L286V mutant cellsmanifested itself in decreased EphB2 expression as judged by RT-PCR(FIG. 6 E, lane 3). Furthermore, addition of 10 μM Compound D led toincreased EphB2 message (FIG. 6 E, lane 4) as well as EphB2 expressionin these cells (FIG. 6 D). These results are consistent with the data ofBatlle and colleagues (Batlle et al., Cell 111, 251 (2002)) who recentlyshowed that expression of EphB2/EphB3 receptors and their ligandephrin-B1 is inversely controlled in colonic crypts via TCF/β-catenintranscription, and that proper regulation is important for appropriatecell proliferation, differentiation and sorting. We present evidencethat the PS-1/L286V mutation via increased TCF/β-catenin signaling,decreased the expression of EphB2 receptors and this is corrected byCompound D mediated inhibition of the β-catenin/CBP interaction.

Example 9 Compound D Causes a G1/S-Phase Arrest and Activates CaspaseActivity

Flow Cytometric Analysis (FACS)

For FACS analysis, approx. 5×10⁶ cells from Compound D-treated orvehicle-treated were fixed with 70% chilled ethanol and stored at −20°C. for at least 30 minutes. The cells were washed once with 1×PBS andincubated with propidium iodine (PI) solution (85 μg/ml propidiumiodine, 0.1% Nonidet P-40, 10 mg/ml RNAse) for 30 minutes at roomtemperature. 10,000 stained cells for each sample were acquired usingBeckman Coulter EPICS XL-MCL Flow Cytometry and the percentage of cellsin different phase of the cell cycle was determined by Expo32 ADCsoftware (Coulter Corporation, Miami, Fla., 33196).

Caspase-3 Activity Assay

SW480, HCT116, and CCD18Co cells were plated at 10⁵ cells per well(96-well plates) for 24 hours prior to treatment. 25 μM of Compound D orcontrol (0.5% DMSO) was added to each well. 24 hours post treatment,cells were lysed and caspase activity was measured using a caspase-3/7activity kit (Apo-One Homogeneous caspase-3/7 assay, #G77905, Promega).Relative fluorescence units (RFU) were obtained by subtracting the unitvalues of the blank (control, without cells) from the experimentalmeasured values.

Compound D Causes a G₁/S-Phase Arrest and Activates Caspase Activity

It has been shown that inhibition of the expression of the cyclin D1gene causes arrest at the G₁/S-phase of the cell cycle (Shintani et al.,“Infrequent alternations of RB pathway (Rb-p16INK4A-cyclin D1) inadenoid cystic carcinoma of salivary glands,” Anticancer Res. 20:2169-75(2000)). HCT116 (FIG. 7A, upper panel) and SW480 (FIG. 7A, lower panel)cells were treated with Compound D (25 μM) (FIG. 7A, right) or control(0.5% DMSO) (FIG. 7A, left) for 24 hours. The cells were subsequentlystained with propidium iodide (PI) and analyzed for DNA content by FACScytofluorometry. As expected, the control cells, (FIG. 7A, left), werecycling normally whereas the Compound D treated cells (FIG. 7A, right)showed increased accumulation at G₁/S-phase of the cell cycle. Thus, itcan be seen that Compound D causes arrest of cells at the G₁ phase.

Caspases are cysteine proteases that are generally activated in a givenpopulation of cells triggered by apoptotic stimuli. To assess apoptoticinduction in SW480, HCT116, and wild-type colonocytes (CCD18Co cells),the cells were treated with either Compound D (25 μM) or control (0.5%DMSO) for 24 hours, followed by an assay for caspase-3/7 activity. Asshown in FIG. 7B, Compound D specifically and significantly activatedthe caspase-3/7 pathway in SW480 and HCT116 cells compared to CCD18Cocells.

Example 10 Compound D Reduces Proliferation of Transformed ColorectalCells

Soft Agar Assays

The soft agar colony formation assay was conducted with SW480 cells bysome modification of the procedure previously described (Moody et al.,“A vasoactive intestinal peptide antagonist inhibits non-small cell lungcancer growth,” Proc. Natl. Acad. Sci. USA. 90:4345-49 (1993)).

Each well (35 mm) of a 6-well plate (Nalge Nunc International, Roskide,Denmark) was coated with 1 ml of 0.8% bottom agar in DMEM mediumcontaining 10% fetal bovine serum. After it was solidified, 1 ml of DMEMmedium containing 0.4% top agar, 10% fetal bovine serum, compound doublyconcentrated, and 5,000 single viable cells was added to each well. Thecultures were incubated at 37° C. in humidified 5% CO₂ incubator.Colonies in soft agar were monitored daily and photographed afterincubation for 8 days. Colonies>60 μm in diameter were counted.

Compound D Reduces Proliferation of Transformed Colorectal Cells

Soft agar colony forming assays were performed using SW480 cells treatedwith Compound D (0.25-5 μM) and 5-fluorouracil (5-FU) (0.5-32 μM). Asshown in FIG. 8A, Compound D shows a dose dependent decrease in thenumber of colonies formed. IC₅₀ value of Compound D and 5-FU was0.87±0.11 μM and 1.98±0.17 μM, respectively. Thus, Compound D increasedcaspase activity and reduced growth in vitro of colorectal cells thatare transformed by mutations that activate β-catenin signaling.

Example 11 Compound C Reduces Tumor Growth in Nude Mouse Model

SW620 cells (9×10⁶ cells/mouse) were grafted into nude micesubcutaneously on Day 0. Mice received 200 mg/kg of Compound Cintraperitoneally every other day until Day 21 after 4 times of 300mg/kg every other day starting Day 1. Compound C reduces the tumorgrowth in the treated mice compared to the vehicle control mice (FIG.9A), and slightly reduces body weights of the treated mice compared tothose of the vehicle control mice (FIG. 9B).

Example 12 Compound D Suprresses Survivin Expression

The effect of Compound D on survivin expression was studied at bothtranscriptional and translational levels. The methods used at thetranscriptional level include cDNA microarray analysis, RT-PCR, survivinreporter assays and chromotin immunoprecipitation (ChIP). The methodsused at translational levels include Western blot analysis andimmunochemistry.

A plasmid containing luciferase under the control of survivin promoterwas constructed and transfected into wild type, CBP+/−, or p300+/−3T3cells. The results (FIG. 10) show that Wnt 1 stimulates expression ofthe survivin gene in all three types of cells, whereas Compound Dreduces expression of the survivin gene and decreases the stimulation ofthe survinin gene expression by Wnt1 in those cells. Similarly, CompoundD and its analog (Comound A) were shown to inhibit expression ofsurvivin in SW480 cells (FIG. 11).

Real time reverse transcription-PCR analysis was performed according tothe protocol provided with the SYBR Green PCR Master Mix Kit (PerkinElmer Biosystems, Shelton, ST). Total RNA templates for the RT-PCRreactions were extracted with the RNeasy Midi Kit (Qiagen) from cellstreated with Compound D (25 μM) or control (0.5% DMSO) 24 hours aftertreatment. The primers used for the RT-PCR reactions were5′-AGCCCTTTCTCAAGGACCAC-3′ and 5′-GCACTTTCTTCGCAGTTTCC-3′. Table 8 showsthe results of the analysis. A ratio less than 0.5 indicates asignificant decrease of gene expression due to the treatment of CompoundD, whereas a ratio greater than 1.5 indicates a significant increase ofgene expression. A ratio about 1 indicates no change. As indicated inTable 8 and FIG. 12, the expression of the survivin gene issignificantly reduced in the presence of Compound D compared to thecontrol.

TABLE 8 Gene Expression with and without Compound D Ratio (Treated/DMSOGene Control) Ubiquitin 0.98 GADPH 0.98 HLAC 1.01 Survivin 0.30 PONA0.33 Antigen KI-67 0.45 MIC-1 7.0 GADD-153 7.00

ChIP assays on SW 480 cells treated with either Compound D (25 μM) orcontrol (0.5% DMSO) were performed. As shown in FIG. 13, the survivinpromoter is occuried by CBP, β-catenin, Tcf4 and acetylated histone incontrol treated cells. Treatment with Compound D decreases theassociation of all these proteins with the survivin promoter.

To characterize the effect of Compound D on the survivin expression atthe translational level, Western blot analysis of extracts of cellstreated with vehicle (0.5% DMSO) alone, 10 μM or 25 μM Compound D, or 5μM 5-FU was performed using survivin 6E4 monoclonal antibody (CellSignaling Technolgy). The results (FIG. 14A) show that the treatmentswith Compound D at both concentrations and the treatment with 5-FUreduced the amount of the survivin protein. The treatments with CompoundD at both concentrations were more effective in reducing the survivinexpression than the treatment with 5-FU, and the treatment with CompoundD at the higher concentration (i.e., 25 μM) was most effective.

The effect of Compound D on the survivin expression at the translationallevel was further characterized using immunofluorescence microscopy. Inthe absence of Compound D, survivin localizes to the mitotic spindleapparatus, consistent with the notion that survivin is involved inchromosomal separation (FIG. 14B). This expression pattern was notobserved in SW480 cells after the treatment of Compound D as little orno survivin protein was detected (FIG. 14C).

Example 13 Effects of Various Compounds on Survivin and TCF4 Expression

The effects of various compounds having general formula (I) on survivinand TCF4 expression were characterized. The results are shown in Table9.

TABLE 9 Effects of compounds on survivin and TCF4 expression Survivin %inhibition TCF4 IC50 5 uM 25 uM (uM)

100 99 ~2

97 100 ~2.2

51 93 ~6.3

41 92 5.2 ± 0.7

0 6 18.2 ± 2.4 

0 80 1.3 ± 0.1

0 93 2.2 ± 0.2

46 96 4.4 ± 0.6

0 77 3.5 ± 0.3

0 92 7.3 ± 0.6

79 81 1.7 ± 0.2

0 84 4.8 ± 0.4

0 68 10.9 ± 1.3 

8 4 NA

9 91 1.4 ± 0.2

5 91  6.3 ± 0.431

0 94 2.6 ± 0.4

0 21 7.3 ± 1.1

0 91 5.2 ± 1.1

45 88 13.2 ± 4.1 

9 92 5.9 ± 0.5

6 58 11.2 ± 1.5 

48 96  3.9 ± 0.55

0 32 50.4 ± 7.0 

86 91 2.6 ± 0.6

27 98 10.7 ± 1.7 

80 97 4.6 ± 0.7

82 97 2.8 ± 0.4

6 89 13.9 ± 2.3 

14 99 10.7 ± 1.9 

25 44 27.1 ± 4.6 

Example 14 Compound D Promotes Apoptosis Via Suppression of SurvivinExpression

To determine the effect of Compound D on apoptosis and the role ofsurvivin in such an effect, the activities of caspases 2 and 3 incultured tumor cells treated with either Compound D or control weremeasured. The results (FIG. 15) show that (1) Compound D (at 2.5 μM or5.0 μM) activated the caspase 3 activity, but not the caspase 2activity; (2) stausporine (0.5 μM) increased both the caspase 2 andcaspase 3 activities; (3) the co-treatment of stausporine and Compound Dproduced a synergic stimuation of the caspase 3 activity, but not asynergic stimuation of the caspase 2 activity; and (4) transfection ofthe survivin gene decreased the activiation of the caspase 3 activityinduced by the treatment of stausporine or Compound D, and the synergicstimulation of the caspase 3 activity induced by the co-treatment ofstausporine and Compound D. The above results suggest that Compound Dstimulate the caspase 3 activity via suppression of the expression ofthe survivin gene.

The effect of compound D on apoptosis and the role of survivin in suchan effect were further characterized by measuring cell death of culturedtumor cells treated with staurosporine (0.5 μM), Compound D (5.0 μM) orboth. The results (FIG. 16) showed that both Compound D and stausporinepromote cell death, and that transfection of the survivin gene decreasedthe increase in cell death induced by the treatment of stausporine,Compound D, or both. The above results suggest that Compound D promoteapoptosis via suppression of the expression of the survivin gene.

To determine the effect of Compound D on cell cycle and the role ofsurvivin in such an effect, FACS analysis was performed on culturedtumor cells with or without transfection of a construct containing thesurvivin gene and further treated with stausporine (0.5 μM), Compound D(5 μM), or both. The results (FIG. 17) show that both stausporine andCompound D increase the number of cells in G₀, and that overexpressionof survivin in the cells decreases the effect of the treatment ofstausporine, Compound D, or both. These results suggest that the effectof Compound D on cell cycle may be at least partially via suppression ofthe expression of the survivin gene.

Example 15 Preparation and Activity of Prodrugs

(1) General Procedure for Preparing Prodrugs by Phosphorylation ofPhenol Group

The starting phenol (26.06 mmol) was dissolved in tetrahydrofuran (130ml), followed by addition of triethylamine (TEA) (10.9 ml, 78.18 mmol)at room temperature. The reaction mixture was cooled to 5° C., and thenPOCl₃ (12.2 ml, 130.3 mmol) was added slowly. After addition wasfinished, the mixture was allowed to warm to room temperature, andstirred at this temperature for 5 hours. After the reaction wascompleted, the mixture was poured into celite-pad filter funnel toremove TEA-HCl salt. Organics was diluted with water (130 ml) at 5° C.,followed by adjusting pH 7˜8 using sodium bicarbonate (50 g), and theresulting basic solution was stirred overnight at room temperature. Theresulting aqueous layer was washed with EtOAc (100 ml), and thenlyophilized. The crude product was dissolved in methylene chloride (100ml), followed by for 1 hour at room temperature. Inorganic salts wereremoved by filtration using celite pad, then solvent was evaporated. Thecrude product was purified by recrystallization (EA/Ether) to get 9.5 gof phosphorylated product as an off-white solid.

(2) Typical Work-up Procedure for the Free Form of Phosphate

After washing the resulting basic aqueous layer, the solution wasacidified to pH 3˜4 using 1 N HCl, and then the phosphate free form wasextracted twice with chloroform (300 ml). The organic layer was driedover sodium sulfate, and the crude product was purified byrecrystallization.

(3) Converting Method from Free Form to Di-sodium Form

A. Titration Method

Free form of phosphate can be transformed to di-sodium salt form bytitration, which could use many inorganic bases. For example, sodiumcarbonate, sodium bicarbonate, and sodium hydroxide are used in thisexperiment to produce di-sodium form. Other cations can be used to makedifferent di-salt forms.

1. Analytical Method and Instrument for Titration

-   -   a. Instrument: TitraLab (RADIOMETER COPENHAGEN)    -   Electrode: pHG201 pH glass electrode    -   (RADIOMETER COPENHAGEN, 945-462)    -   REF201 reference electrode with KCl salt-bridge solution    -   (RADIOMETER COPENHAGEN, 945-463)    -   Titrant: 10 M Na₂CO₃    -   Burette speed (titration speed): 15% (=1.5 ml/min)    -   Sample: 50 mg dissolved in distilled water (30 ml)    -   b. Results    -   pH 4 (start pH=2)

EP1 EP2 n start pH pH Titrant (ml) pH Titrant (ml) 1 2.10 4.21 9.50 8.1519.03 2 2.08 4.26 10.28 8.02 19.12 Mean 2.09 4.24 9.89 8.09 19.08B. Using Organic Sodium Donor

The basic drawback of titration using inorganic base is that the watermust be used for the solvent. So searching the sodium donor dissolvedfreely in normal organic solvent is the easiest way to solve theproblem. Several reagents such as sodium acetate and sodiumethylhexanoic acid were tested and found to be useful for making adi-sodium salt form.

Table 10 shows compounds for bioactivity test selected from the prodrugsof the present invention and IC₅₀ values thereof, which are measured bythe reporter gene assay (RGA) and oncogenic activity by MTS orSulforhodamine B assay as described in Example 6. The compound numberson Table 10 are unrelated to those in Table 4 or 5.

TABLE 10 THE REPORTER GENE ASSAY AND ONCOGENIC ACTIVITY BY MTS ORSULFORHODAMINE B ASSAY FOR SELECTED PRODRUG COMPOUNDS Assay RGA, RGA,TopF Survivin MTS, SW480 MTS, HCT116 IC50, IC50, (uM) (uM) No StructureuM uM LD50 GI50 LD50 GI50 1

4.2 6.4 17.0 2.0 16.1 2.2 2

3.5 5.7  8.2 3.1 23.2 6.6 3

11.5 ND up to 50 uM 3.0 41.9 3.1 4

7.3 6.5 ND up to 50 uM 6.9 49.3 11.4 5

26.0 34.0 5.2 ND up to 50 uM 16.5 6

0.8 0.1  9.2 0.5  6.4 0.4 7

2.3 1.0 12.9 2.2 12.0 1.8 8

1.4 0.9 21.6 2.1 23.2 1.9 9

9.6 6.0 ND up to 50 uM 7.6 ND up to 50 uM 14.7 10

2.8 1.7  9.4 0.9  7.9 0.8 11

10.3 6.7 ND up to 50 uM 6.5 ND up to 50 uM 6.3 12

1.0 0.7 ND up to 50 uM 1.0 19.3 1.2 13

1.8 0.9 21.1 2.3 20.0 1.7 14

1.7 1.2 21.1 2.3 16.0 2.1

Example 16 Solubility of Selected Prodrugs

General Procedure for Solubility Test of Prodrugs

About 2 mg of each prodrug was dissolved in 1 ml of JP1 or JP2 solutionas indicated below. Incubating at a temperature of 37° C., 200 ul ofsamples were withdrawn at 0 hour, 2 hour and 20 hour. Withdrawn sampleswere filtered through 0.45 μm syringe filters and analyzed by HPLCsystem.

Composition of Artificial Gastro-Intestinal Fluids (JP1, JP2)

JP1 JP2 PH  1.2 pH 6.8 NaCI  2.0 g 0.2 M KH₂PO₄ 250 ml 10% HCI 24.0 ml0.2N NaOH 118 ml Distilled H₂O Adjusted to 1 L Distilled H₂O Adjusted to1 L

Table 11 below shows the results of solubility test of selectedprodrugs. The compound numbers on Table 11 are unrelated to those inTable 4, 5 or 10.

TABLE 11 AQUEOUS SOLUBILITY FOR SELECTED PRODRUG COMPOUNDS Solubility(37° C., ug/mL) 0 hr, 2 hr, 20 hr No Structure JP1 (pH 1.2) JP2 (pH 6.8)1

60.1 87.3 92.8 1797 1867 1894 2

122 173 160 1950 1939 1940 3

1878 1971 2036 1325 1902 2005 4

554 646 756 1982 2014 2030 5

406 532 684 1761 1778 1758 6

1453 1724 1787 1829 1864 1867 7

309 446 521 2145 2221 2239 8

671 775 921 2295 2317 2272 9

2251 2275 2403 2322 2353 2421 10

2292 2274 2327 2028 2055 2027 11

2006 2000 1998 1636 1654 1651

Example 17 Preparation of Dimethyl-carbamic acid4-[2-allyl-1benzylcarbamoyl-8-(2,4-difluoro-benzyl)-4,7-dioxo-octahydro-pyrazino[2,1-c][1,2,4]triazin-6-ylmethyl]-phenylester

To a stirred solution of starting material (SM) (8.0 g, 13.9 mmol) andpotassium carbonate (5.8 g, 41.7 mmol) in dimethylformamide was addeddimethylcarbamyl chloride (3.0 g, 27.8 mmol). The reaction mixture wasstirred overnight and then dissolved in EtOAc, washed with water fivetimes. The combined organic layer was washed with brine, dried oversodium sulfate, concentrated in vacuo. The residue was chromatographedon silica gel with neat EtOAc to afford product (4.2 g, 32%). The datafrom analyzing the resulting product by mass spectrometry and NMR are:

MS (ESI): m/e 647 (M+1), 669 (M+Na). ¹H-NMR (300 MHz, CDCl₃) δ 7.27-7.39(6H, m), 6.78-7.13 (6H, m), 6.68 (1H, t, J=6.0 Hz), 5.61-5.70 (2H, m),5.34 (1H, t, J=6.0 Hz), 5.06-5.20 (2H, m), 4.31-4.69 (4H, m), 3.24-3.58(8H, m), 3.07 (3H, s), and 2.99 (3H, s).

Example 18 Preparation of Carbonic acid4-[2-Allyl-1-Benzylcarbamoyl-8-(2,4-difluorobenzyl)-4,7-dioxo-octahydro-pyrazino[2,1-c][1,2,4]triazin-6-ylmethyl]-phenylester 4-nitro-phenyl ester (2)

To a stirred solution of2-Allyl-8-(2,4-difluoro-benzyl)-6-(4-hydroxybenzyl)-4,7-dioxohexahydro-pyrazino[2,1-c][1,2,4]triazine-1-carboxylicacid benzylamine (1) (2 g, 3.47 mmol) in THF (40 ml) was addedTriethylamine (0.97 ml, 6.95 mmol) and 4-nitrophenylchloroformate (0.7g, 3.47 mmol). After stirring at room temperature for overnight, thesolvent was removed under reduced pressure. The crude compound waspurified by chromatography (Hexane, EtOAc 1:1) to give the compound(1.59 g, 62%). The data from analyzing the purified compound by TLCsystem and NMR are:

TLC System: R_(f)=0.3 (n-Hexane:EtOAc 1:1) ¹H NMR (300 MHz, CDCl₃): δ3.25-3.60 (m, 8H), 4.35 (dd, J=14.9 Hz, 5.7 Hz, 1H), 4.43 (dd, J=14.8Hz, 6.1 Hz, 1H), 4.52 (d, 14.5 Hz, 1H), 4.68 (d, 14.1H), 5.10 (d, 17.1Hz, 1H), 5.21 (d, 10.3 Hz, 1H), 5.34 (t, J=6.1 Hz, 1H), 5.58 (dd, J=11.1Hz, 4.1 Hz, 1H), 5.67 (m, 1H), 6.71 (t, J=6.1 Hz, NH), 6.75-6.98 (m,2H), 7.13 (d, J=8.4 Hz, 2H), 7.21 (d, J=8.7 Hz, 2H), 7.23-7.39 (m, 6H),7.46 (d, J=9.5 Hz, 2H), and 8.30 (d, J=9.5 Hz, 2H).

Example 19 Preparation of (2-Dimethylamino-ethyl)-Carbamic acid4-[2-Allyl-1-benzylcarbamoyl-8-(2,4-difluoro-benzyl)-4,7-dioxo-octahydro-pyrazine[2,1-c][1,2,4]triazine-6-ylmethyl]-phenylester hydrochloride salt (3)

To a stirred solution of carbonic acid4-[2-allyl-1-benzylcarbamoyl-8-(2,4-difluoro-benzyl)-4,7-dioxo-octahydro-pyrazino[2,1-c][1,2,4]triazin-6-ylmethyl]-phenylester 4-nitro-phenyl ester (2) (1.2 g, 1.62 mmol) in DMF (25 ml) wasadded N,N-Dimethylethylenediamine (0.26 ml, 2.43 mmol). After stirringat room temperature for overnight, the solvent was removed under reducedpressure. The residue was diluted with EtOAc and washed with water,brine. The organic layer was dried with Na₂SO₄ and concentrated invacuo. The crude compound was purified by chromatography(n-Hexane:EtOAc, 1:1; EtOAc; CH₂Cl₂:MeOH, 9:1).

The compound was poured with water and was maintained pH 5-6 with 1 Naq. HCl to make HCl salt and then lyophilized to get the compound (0.75g, 60%). The data from analyzing the resulting compound by TLC systemand NMR are:

TLC System: R_(f)=0.35 (CH₂Cl₂:MeOH, 9:1) ESI-MS: M+H⁺ 690.39 ¹H NMR(300 MHz, CDCl₃): δ 2.56 (d, J=4.6 Hz, 6H), 3.18 (bm, 4H), 3.41-3.58 (m,4H), 3.75 (t, J=10.3 Hz, 1H), 4.21 (bt, 2H), 4.35 (d, J=14.9 Hz 1H),4.70 (d, J=14.9 Hz 1H), 5.05 (m, 1H), 5.12 (m, 1H), 5.42 (m, 6H), 5.80(m, 1H), 6.91 (d, J=7.6 Hz, 2H), 7.04 (d, J=7.3, 2H), 7.09 (m, 1H),7.18-7.26 (m, 6H), 7.31 (d, J=6.9 Hz, 2H), 7.33 (m, 1H), 7.84 (bt, NH),7.98 (bt, NH), 10.7 (bs, 1H)

Example 20 Mouse In Vivo PK Study of Prodrug a after Single I.V. BolusInjection

Animal Experiment

Drugs were prepared 10 mg/kg/5 ml in 10% Tween 80. Studies wereperformed in ICR mice. After i.v. bolus injection through the tail vein,blood samples were acquired from inferior vena cava at several timepoints and separated to plasma by centrifugation. Plasma samples werepreserved at −20° C. until they were analyzed. Bleeding time points were3, 6, 9, 17, 34, 67, 134, 202, 302 min. N=4.

Sample Preparation

For calibration curve, 98 ul aliquots of control mouse plasma were added2 ul of drug stock solution and added 2 ul internal standard stocksolution, 5 ug/ml of internal standard. Final concentrations ofcalibration samples were 1, 10, 100, 1000 ng/ml and 10 ug/ml. For plasmasample from animal experiment, 100 ul of plasma were added 2 ul ofinternal standard. Then, all the samples were added 500 ul ofacetonitrile, 500 ul of ethylacetate and 100 ul of DW. Samples weremixed for 10 min and centrifuged. The supernatants were transferredanother tubes and evaporated. Adding 200 ul of 40% acetonitrile, theywere reconstituted and analyzed by LC-MS system.

Results

The changes of concentrations of prodrug A and its parent compound inmouse plasma with the increase of time after i.v. bolus injection ofprodrug A are shown in FIG. 18. Square: parent compound; Diamond:prodrug A.

It will be appreciated that, although specific embodiments of theinvention have been described herein for the purposes of illustration,various modifications may be made without departing from the spirit andscope of the invention. Accordingly, the invention is not limited exceptby the appended claims.

All of the above U.S. patents, U.S. patent application publications,U.S. patent applications, foreign patents, foreign patent applicationsand non-patent publications referred to in this specification and/orlisted in the Application Data Sheet, including U.S. patent applicationSer. No. 10/087,443 filed on Mar. 1, 2002, and U.S. patent applicationSer. No. 09/976,470 filed on Oct. 12, 2001, are incorporated herein byreference.

From the foregoing it will be appreciated that, although specificembodiments of the invention have been described herein for purposes ofillustration, various modifications may be made without deviating fromthe spirit and scope of the invention. Accordingly, the invention is notlimited except as by the appended claims.

1. A compound having the following general formula (VII)—Y—R₁₀  (VI) wherein (VI) is the formula:

wherein when not linked to R₁₀, R_(a) is a phenyl group; a substitutedphenyl group having one or more substituents wherein the one or moresubstituents are independently selected from one or more of amino,amidino, guanidino, hydrazino, amidazonyl, C₁₋₄alkylamino,C₁₋₄dialkylamino, halogen, perfluoro C₁₋₄alkyl, C₁₋₄alkyl, C₁₋₃alkoxy,nitro, carboxy, cyano, sulfuryl, and hydroxyl groups; a benzyl group; asubstituted benzyl group with one or more substituents where the one ormore substituents are independently selected from one or more of amino,amidino, guanidino, hydrazino, amidazonyl, C₁₋₄alkylamino,C₁₋₄dialkylamino, halogen, perfluoro C₁₋₄alkyl, C₁₋₃alkoxy, nitro,carboxy, cyano, sulfuryl, and hydroxyl group; or a bicyclic aryl grouphaving 8 to 11 ring members, which may have 1 to 3 heteroatoms selectedfrom nitrogen, oxygen or sulfur; R_(b) is a monocyclic aryl group having5 to 7 ring members, which may have 1 to 2 heteroatoms selected fromnitrogen, oxygen or sulfur, and aryl ring in the compound may have oneor more substituents selected from a group consisting of halide,hydroxy, cyano, lower alkyl, and lower alkoxy groups; Rc is a saturatedor unsaturated C₁₋₆alkyl, C₁₋₆alkoxy, perfluoro C₁₋₆alkyl group; and X₁,X₂, and X₃ may be the same or different and independently selected fromhydrogen, hydroxyl, and halide; and Wherein one of R_(a), R_(b), R_(c),X₁, X₂, and X₃ is linked to R₁₀ via Y, Y is an oxygen, sulfur, ornitrogen in R_(a), R_(b), and R_(c), or an oxygen in X₁, X₂ and X₃; andY—R₁₀ is hemimalate, dimethylaminoalkylcarbamates, or a salt thereof; orR₁₀ is hydroxyalkyl, glycosyl, substituted or unsubstituted piperidineoxycarbonyl, or a salt thereof.
 2. A pharmaceutical compositioncomprising a compound according to claim 1 and pharmaceuticallyacceptable carrier.
 3. The compound of claim 1, wherein when not linkedto R₁₀, R_(a) is a phenyl group; a substituted phenyl group having oneor more substituents wherein the one or more substituents areindependently selected from one or more of amino, amidino, guanidino,hydrazino, amidazonyl, C₁₋₄alkylamino, C₁₋₄dialkylamino, halogen,perfluoro C₁₋₄alkyl, C₁₋₄alkyl, C₁₋₃alkoxy, nitro, carboxy, cyano,sulfuryl, and hydroxyl groups; a benzyl group; a substituted benzylgroup with one or more substituents where the one or more substituentsare independently selected from one or more of amino, amidino,guanidino, hydrazino, amidazonyl, C₁₋₄alkylamino, C₁₋₄dialkylamino,halogen, perfluoro C₁₋₄alkyl, C₁₋₃alkoxy, nitro, carboxy, cyano,sulfuryl, and hydroxyl group; a naphthyl group; a quinolinyl group; anindazolyl group; or a benzpyrazolyl group; an isoquinolinyl group; andR_(b) is phenyl, pyridyl or piperidyl, all of which may be substitutedwith one or more substituents selected from a group consisting ofhalide, hydroxy, cyano, lower alkyl, and lower alkoxy groups.